METHODS AND SYSTEMS FOR PHYLOGENETIC ANALYSIS

CROSS-REFERENCE

[0001] This application is related to and claims priority to the following co-pending U.S. provisional patent applications: U.S. Application Serial No. 61/259,565 [Attorney Docket No. IB-2733P1], filed on November 9, 2009; U.S. Application Serial No. 61/317,644 [Attorney Docket No. IB-2733P2], filed on March 25, 2010; U.S. Application Serial No. 61/347,817 [Attorney Docket No. IB-2733P3], filed on May 24, 2010; U.S. Application Serial No. 61/252,620 [Attorney Docket No. IB-2229P4], filed October 16, 2009; each of which are incorporated herein by reference.

[0002] This application is related to the co-pending international application having application number PCT US2010/040106 [Attorney Docket No. IB-2733PCT], filed on June 25, 2010, which is incorporated herein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with Government support under Contract No. DE-AC02-05CH11231 awarded by the Department of Energy; a grant from the Department of Homeland Security and Agreement Number 07-576-550-0 from State of California Water Quality Board, and a grant from the National Institutes of Health having Award Number AI075410. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0004] With as many as 10 ³⁰ microbial genomes globally, across multiple different environmental and host conditions, variety both within and between microbiomes is well recognized (Huse et al. (2008), PLoS Genetics 4(11): el000255). As a result of this variety, characterizing the contents of a microbiome is a challenge for current approaches. Firstly, standard culturing techniques are successful in maintaining only a small fraction of the microorganisms in nature. Means of more direct profiling, such as sequencing, face two additional challenges. Both the sheer number of different genomes in a given sample and the degree of homology between members present a complex problem for already laborious procedures.

[0005] Biopolymers such as nucleic acids and proteins are often identified in the search for useful genes, to diagnose diseases or to identify organisms. Frequently, hybridization or another binding reaction is used as part of the identification step. As the number of possible targets increases in a sample, the design of systems to detect the different hybridization reactions increases in difficulty along with the analysis of the binding or hybridization data. The design and analysis problems become acute when there are many similar targets in a sample as is the case when the individual species or groups that comprise a microbiome are detected or quantified in a single assay based on a highly conserved polynucleotide. For example, while approximately 98% of bacteria found in the human gut belong to only four bacterial divisions, this includes approximately 36,000 different phylotypes at the strain level, having >99% sequence identity (Hattori et al. (2009), DNA Res. 16: 1-12). While possibly containing certain overlapping taxa, the different environments presented by the guts of other hosts are expected to support different microbiomes. In situations where contributions from multiple sub-enviroments are combined, such as a water source potentially contaminated by a variety of sources, just identifying the thousands of taxa is a significant challenge to current methods of detection.

[0006] Since the study of microbiomes can offer new insight into origins of environmental change, disease, immunological functions, and physiological functions, improved methods for designing nucleic acids, proteins, or other probes that can recognize specific organisms, or taxa are needed. Similarly, improved methods for data analysis that allow detection and quantification of the members of a microbial community at high confidence levels are also needed.

SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides a method for determining a pulmonary condition of a subject. In one embodiment, the method comprises: (a) contacting a sample from said subject with a plurality of different probes; (b) determining hybridization signal strength for each of said probes, wherein said determination establishes a biosignature for said sample; and, (c) determining a pulmonary condition of said subject based on the results of step (b). In some embodiments, step (b) further comprises comparing the biosignature of said sample to a biosignature for one or more pulmonary conditions. In some embodiments, the sample is a pulmonary sample, including but not limited to sputum, endotracheal aspirate, a bronchoalveolar lavage sample, or a swab of the endotrachea. In some embodiments, the method further comprises making a healthcare decision based on the results of step (c). In some embodiments, the biosignature comprises the presence, relative abundance, and/or quantity of one or more OTUs selected from OTUs listed in one or more of Table 3, Table 4, or Table 5. In some embodiments, the biosignature comprises the presence, relative abundance, and/or quantity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or more OTUs listed in one or more of Table 3, Table 4, or Table 5. In some embodiments, the pulmonary condition is selected from the group consisting of: healthy, exacerbated COPD, non-exacerbated COPD, and intermediate COPD exacerbation, wherein intermediate COPD exacerbation comprises a prediction of the onset of exacerbation of COPD in said subject. In some embodiments,

[0008] In one aspect, the invention provides a method of classification, diagnosis, prognosis, and/or prediction of an outcome of a pulmonary condition in a subject. In one embodiment, the method comprises: (a) isolating nucleic acid material from a sample from said subject; (b) determining hybridization signal strength distributions of negative control probes that do not specifically hybridize to one or more highly conserved polynucleotides in one or more target operational taxon units (OTUs); (c) determining hybridization signal strengths for a plurality of different interrogation probes, each of which is complementary to a section within said one or more highly conserved polynucleotides; (d) using the hybridization signal strengths of the negative and positive probes to determine the probability that the hybridization signal for the different interrogation probes represents the presence, relative abundance, and/or quantity of said one or more OTUs; and, (e) classifying, diagnosing, prognosing, and/or predicting an outcome of said pulmonary condition based on the results of step (d). In some embodiments, the sample is a pulmonary sample, including but not limited to sputum, endotracheal aspirate, a bronchoalveolar lavage sample, or a swab of the endotrachea. In some embodiments, the method further comprises making a healthcare decision based on the results of step (e). In some embodiments, the pulmonary condition is selected from the group consisting of: healthy, exacerbated COPD, non- exacerbated COPD, and intermediate COPD exacerbation, wherein intermediate COPD exacerbation comprises a prediction of the onset of exacerbation of COPD in said subject. In some embodiments, the presence, relative abundance, and/or quantity is detected with a confidence level greater than 95%. Highly conserved polynucleotides include, but are not limited to, 16S rRNA gene, 23 S rR A gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, nifl3 gene, RNA molecules derived therefrom, or a combination thereof.

[0009] In one aspect, the invention provides a method for assessing a pulmonary condition of a subject. In one embodiment, the method comprises detecting in a sample from said subject the presence, relative abundance, and/or quantity of one or more OTUs in a single assay, wherein said one or more OTUs are selected from OTUs listed in one or more of Table 3, Table 4, or Table 5; and determining the pulmonary condition of said subject based on said detection. In some embodiments, the presence, relative abundance, and/or quantity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 100, 250, 300, 400, 500, 600, 700, 800, 900, 1000, or more OTUs listed in one or more of Table 3, Table 4, or Table 5 are detected in a single assay. In some embodiments, the sample is a pulmonary sample, including but not limited to sputum, endotracheal aspirate, a bronchoalveolar lavage sample, or a swab of the endotrachea. In some embodiments, the method further comprises making a healthcare decision based on the determination of the pulmonary condition of the subject. In some embodiments, the pulmonary condition is selected from the group consisting of: healthy, exacerbated COPD, non- - exacerbated COPD, and intermediate COPD exacerbation, wherein intermediate COPD exacerbation comprises a prediction of the onset of exacerbation of COPD in said subject. In some embodiments, the presence, relative abundance, and/or quantity is detected with a confidence level greater than 95%.

[0010] In one aspect, the invention provides a system for practicing the methods of the invention. In one embodiment, the system comprises: (a) negative control probes that do not specifically hybridize to one or more highly conserved polynucleotides in a plurality of target OTUs; and (b) a plurality of different interrogation probes, each of which is complementary to a section within said one or more highly conserved polynucleotides in one or more of said plurality of target OTUs, wherein said plurality of target OTUs consists of OTUs in one or more of Table 3, Table 4, or Table 5. Highly conserved polynucleotides include, but are not limited to, 16S rR A gene, 23S rR A gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, nifl3 gene, RNA molecules derived therefrom, or a combination thereof. In some embodiments, the system further comprises a plurality of positive control probes, such as probes comprising sequences selected from SEQ ID NOs: 51-100, and/or the complements thereof.

[0011] Probes used in methods and systems of the present invention can be used to detect the presence, absence, relative abundance, and/or quantity of at least 10,000 different OTUs in a single assay. In some embodiments, probes are attached to a substrate. Substrates can comprise any suitable material, including but not limited to glass, plastic, or silicon. Substrates can take any suitable shape, such as a flat surface, a bead, or a microsphere.

INCORPORATION BY REFERENCE

[0012] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0014] Figure 1 illustrates an example of a suitable computer system environment.

[0015] Figure 2 illustrates a networked system for the remote acquisition or analysis of data obtained through a method of the invention.

[0016] Figure 3 illustrates a flow chart of the probe selection process.

[0017] Figures 4A-B demonstrate the distribution of observed pair difference score, d, from quantitative standards (QS) probes and negative controls (NC) probes.

[0018] Figure 5 is a graph showing variations of gamma scale across 79 arrays.

[0019] Figure 6 illustrates the pre-partition process for computational load balancing.

[0020] Figure 7 is a chart showing the concentration of 16S amplicon versus PhyloChip response.

[0021] Figure 8 is boxplot comparison of the detection algorithm based on pair "response score",r, distribution (novel) versus the positive fraction calculation (previously used with the G2 PhyloChip.

[0022] Figure 9 is two graphs that show the comparison of the r score metric versus the pf by receiver operator characteristic (R.O.C) plots. [0023] Figure 10A illustrates a phylogenetic tree exhibiting family level bacterial diversity detected in COPD airways following antimicrobial administration.

[0024] Figure 10B illustrates bacterial richness detected in individual patient samples.

[0025] Figure 11 illustrates the results of NMDS analysis showing bacterial community composition that is highly influenced by the duration of intubation, with subjects COPD 5 and COPD 6 superimposed on the right side of the figure, indicative of highly similar bacterial community composition.

[0026] Figure 12 shows a phylogenetic tree illustrating core bacterial taxa detected in COPD airways sample, with known pathogens denoted with an asterisk, and distinct bacterial families indicated by different shades of gray.

[0027] Figure 13 illustrates the time points of 25 sputa samples before, during, and after clinical exacerbation of COPD, with lines for subjects 3, 19, and 49 having exacerbations clinically considered to be infectious related, first and second time points pre-exacerbation designated prel and pre2, respectively, and first and second time points post-exacerbation are designated postl and post2, respectively.

[0028] Figure 14 a graph that illustrates bacterial community richness as measured by 16S rRNA PhyloChip analysis.

[0029] Figure 15 is a graph showing bacterial community richness over time, with sampling time points combined across subjects.

[0030] Figure 16 illustrates bacterial diversity overtime for each subject as determined by 16S rRNA PhyloChip analysis, using an inverse Simpson index.

[0031] Figure 17 is a graph showing bacterial community diversity over time, with inverse Simpson indices for each time point combined across subjects.

[0032] Figure 18 is a graph showing bacterial community diversity over time, with Shannon indices for each time point combined across subjects.

[0033] Figure 20 illustrates a hierarchical cluster analysis of bacterial community composition across samples based on a Bray-Curtis distance metric of dissimilarities in community composition.

[0034] Figure 21 illustrates an ordination-based analysis of the variation in bacterial community composition across subject samples, using non-metric multidimensional scaling (NMDS), where each circle represents the total bacterial community present in that sample.

[0035] Figure 22 illustrates changes in relative abundance from time points prel to exacerbation for selected taxa from subject 3, where each bar is a distinct taxon, a positive change indicates increased relative abundance at the later time point, and a negative change indicates decreased relative abundance at the later time point.

[0036] Figure 23 illustrates changes in relative abundance from time points exacerbation to post2 for selected taxa from subject 3, where each bar is a distinct taxon, a positive change indicates increased relative abundance at the later time point, and a negative change indicates decreased relative abundance at the later time point. [0037] Figure 24 illustrates changes in relative abundance from each time point to the next for selected taxa from subject 19, where each bar is a distinct taxon, a positive change indicates increased relative abundance at the later time point, and a negative change indicates decreased relative abundance at the later time point.

[0038] Figure 25 illustrates changes in relative abundance from time points prel to pre2 and from pre2 to exacerbation for selected taxa from subject 49, where each bar is a distinct taxon, a positive change indicates increased relative abundance at the later time point, and a negative change indicates decreased relative abundance at the later time point.

[0039] Figure 26 illustrates changes in relative abundance from time points exacerbation (Exac) to postl and from postl to post2 for selected taxa from subject 49, where each bar is a distinct taxon, a positive change indicates increased relative abundance at the later time point, and a negative change indicates decreased relative abundance at the later time point.

[0040] Figure 27 illustrates changes in relative abundance from each time point to the next for selected taxa from subject 40, where each bar is a distinct taxon, a positive change indicates increased relative abundance at the later time point, and a negative change indicates decreased relative abundance at the later time point.

[0041] Figure 28 illustrates changes in relative abundance from each time point to the next for selected taxa from subject 46, where each bar is a distinct taxon, a positive change indicates increased relative abundance at the later time point, and a negative change indicates decreased relative abundance at the later time point.

[0042] Figure 29 illustrates the bacterial community distribution at the family level for subject 3 over time.

[0043] Figure 30 illustrates the bacterial community distribution at the class level for subject 3 over time.

[0044] Figure 31 illustrates the bacterial community distribution at the family level for subject 19 over time.

[0045] Figure 32 illustrates the bacterial community distribution at the class level for subject 19 over time.

[0046] Figure 33 illustrates the bacterial community distribution at the family level for subject 49 over time.

[0047] Figure 34 illustrates the bacterial community distribution at the class level for subject 49 over time.

[0048] Figure 35 illustrates the bacterial community distribution at the family level for subject 40 over time.

[0049] Figure 36 illustrates the bacterial community distribution at the class level for subject 40 over time. [0050] Figure 37 illustrates the bacterial community distribution at the family level for subject 46 over time.

[0051] Figure 38 illustrates the bacterial community distribution at the class level for subject 46 over time.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0052] As used herein, the term "oligonucleotide" refers to a polynucleotide, usually single stranded, that is either a synthetic polynucleotide or a naturally occurring polynucleotide. The length of an oligonucleotide is generally governed by the particular role thereof, such as, for example, probe, primer and the like. Various techniques can be employed for preparing an oligonucleotide, for instance, biological synthesis or chemical synthesis. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10): 1925 (1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al., Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984), Letsinger, et al., J. Am. Chem. Soc, 110:4470 (1988); and Pauwels, et al., Chemica Scripta, 26: 141 (1986)); phosphorothioate (Mag, et al, Nucleic Acids Res., 19: 1437 (1991); and U.S. Pat. No. 5,644,048); phosphorodithioate (Briu, et al., J. Am. Chem. Soc, 111:2321 (1989)); 0- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc, 114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl., 31 : 1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207 (1996), all of which are incorporated by reference)). Other analog nucleic acids include those with positive backbones (Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionic backbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English, 30:423 (1991); Letsinger, et al., J. Am. Chem. Soc, 110:4470 (1988); Letsinger, et al., Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al., Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J. Biomolecular NMR, 34: 17 (1994); Tetrahedron Lett., 37:743 (1996)); and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins, et al., Chem. Soc Rev., (1995) pp. 169-176). Several nucleic acid analogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. All of these references are hereby expressly incorporated by reference.

[0053] The nucleic acid may be DNA, RNA, or a hybrid and may contain any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthanine, hypoxanthanine, isocytosine, isoguanine, and base analogs such as nitropyrrole and nitroindole, etc. Oligonucleotides can be synthesized by standard methods such as those used in commercial automated nucleic acid synthesizers and later attached to an array, bead or other suitable surface. Alternatively, the oligonucleotides can be synthesized directly on the assay surface using photolithographic or other techniques. In some embodiments, linkers are used to attach the oligonucleotides to an array surface or to beads.

[0054] As used herein, the term "nucleic acid molecule" or "polynucleotide" refers to a compound or composition that is a polymeric nucleotide or nucleic acid polymer. The nucleic acid molecule may be a natural compound or a synthetic compound. The nucleic acid molecule can have from about 2 to 5,000,000 or more nucleotides. The larger nucleic acid molecules are generally found in the natural state. In an isolated state, the nucleic acid molecule can have about 10 to 50,000 or more nucleotides, usually about 100 to 20,000 nucleotides. It is thus obvious that isolation of a nucleic acid molecule from the natural state often results in fragmentation. It may be useful to fragment longer target nucleic acid molecules, particularly RNA, prior to hybridization to reduce competing intramolecular structures. Fragmentation can be achieved chemically, enzymatically, or mechanically. Typically, when the sample contains DNA, a nuclease such as deoxyribonuclease (DNase) is employed to cleave the phosphodiester linkages. Nucleic acid molecules, and fragments thereof, include, but are not limited to, purified or unpurified forms of DNA (dsDNA and ssDNA) and RNA, including tRNA, mRNA, rRNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA/RNA hybrids, biological material or mixtures thereof, genes, chromosomes, plasmids, cosmids, the genomes of microorganisms, e.g., bacteria, yeasts, phage, chromosomes, viruses, viroids, molds, fungi, or other higher organisms such as plants, fish, birds, animals, humans, and the like. The polynucleotide can be only a minor fraction of a complex mixture such as a biological sample.

[0055] As used herein, the term "hybridize" refers to the process by which single strands of polynucleotides form a double-stranded structure through hydrogen bonding between the constituent bases. The ability of two polynucleotides to hybridize with each other is based on the degree of complementarity of the two polynucleotides, which in turn is based on the fraction of matched complementary nucleotide pairs. The more nucleotides in a given polynucleotide that are complementary to another polynucleotide, the more stringent the conditions can be for hybridization and the more specific will be the binding between the two polynucleotides. Increased stringency may be achieved by elevating the temperature, increasing the ratio of co-solvents, lowering the salt concentration, and combinations thereof. [0056] As used herein, the terms "complementary," "complement," and "complementary nucleic acid sequence" refer to the nucleic acid strand that is related to the base sequence in another nucleic acid strand by the Watson-Crick base-pairing rules. In general, two polynucleotides are complementary when one polynucleotide can bind another polynucleotide in an anti-parallel sense wherein the 3'-end of each polynucleotide binds to the 5'-end of the other polynucleotide and each A, T(U), G, and C of one polynucleotide is then aligned with a T(U), A, C, and G, respectively, of the other polynucleotide. Polynucleotides that comprise R A bases can also include complementary G/U or U/G basepairs. Two complementary strands may comprise complementary regions comprising all or one or more portions of one or both strands.

[0057] As used herein, the term "clustering tree" refers to a hierarchical tree structure in which observations, such as organisms, genes, and polynucleotides, are separated into one or more clusters. The root node of a clustering tree consists of a single cluster containing all observations, and the leaf nodes correspond to individual observations. A clustering tree can be constructed on the basis of a variety of characteristics of the observations, such as sequences of the genes and morphological traits of the organisms. Many techniques known in the art, e.g. hierarchical clustering analysis, can be used to construct a clustering tree. A non- limiting example of the clustering tree is a phylogenetic, taxonomic or evolutionary tree.

[0058] As used herein, the terms "operational taxon unit," "OTU," "taxon," "hierarchical cluster," and "cluster" are used interchangeably. An operational taxon unit (OTU) refers to a group of one or more organisms that comprises a node in a clustering tree. The level of a cluster is determined by its hierarchical order. In one embodiment, an OTU is a group tentatively assumed to be a valid taxon for purposes of phylogenetic analysis. In another embodiment, an OTU is any of the extant taxonomic units under study. In yet another embodiment, an OTU is given a name and a rank. For example, an OTU can represent a domain, a sub-domain, a kingdom, a sub-kingdom, a phylum, a sub-phylum, a class, a sub^class, an order, a sub-order, a family, a subfamily, a genus, a subgenus, or a species. In some embodiments, OTUs can represent one or more organisms from the kingdoms eubacteria, protista, or fungi at any level of a hierarchal order. In some embodiments, an OTU represents a prokaryotic or fungal order.

[0059] As used herein, the term "kmer" refers to a polynucleotide of length k. In some embodiments, k is an integer from 1 to 1000. In some embodiments, k is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000.

[0060] As used herein, the term "perfect match probe" (PM probe) refers to a kmer which is 100% complementary to at least a portion of a highly conserved target gene or polynucleotide. The perfect complementarity usually exists throughout the length of the probe. Perfect probes, however, may have a segment or segments of perfect complementarity that is/are flanked by leading or trailing sequences lacking complementarity to the target gene or polynucleotide.

[0061] As used herein, the term "mismatch probe" (MM probe) refers a control probe that is identical to a corresponding PM probe at all positions except for one, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of the PM probe. Typically, the non-identical position or positions are located at or near the center of the PM probe. In some embodiments, the mismatch probes are universal mismatch probes, e.g., a collection of mismatch probes that have no more than a set number of nucleotide variations or substitutions compared to positive probes. For example, the universal mismatch probes may differ in nucleotide sequence by no more than five nucleotides compared to any one PM probe in the PM probe set. In some embodiments, a MM probe is used adjacent to each test probe, e.g., a PM probe targeting a bacterial 16S rRNA sequence, in the array.

[0062] As used herein, the term "probe pair" refers to a PM probe and its corresponding MM probe. In some embodiments, the PM probes and the MM probes are scored in relation to each other during data processing and statistic analysis. As used herein, the term "a probe pair associated with an OTU" is defined as a pair of probes consisting of an OTU-specific PM probe and its corresponding MM probe.

[0063] As used herein, a "sample" is from any source, including, but not limited to a biological sample, a gas sample, a fluid sample, a solid sample, or any mixture thereof

[0064] As used herein, a "microorganism" or "organism" includes, but is not limited to, a virus, viroids, bacteria, archaea, fungi, protozoa and the like.

[0065] The term "sensitivity" refers to a measure of the proportion of actual positives which are correctly identified as such.

[0066] The term "specificity" refers to a measure of the proportion of actual negatives which are correctly identified as such.

[0067] The term "confidence level" refers to the likelihood, expressed as a percentage, that the results of a test are real and repeatable, and not random. Confidence levels are used to indicate the reliability of an estimate and can be calculated by a variety of methods.

Biosignatures

[0068] In one aspect, the invention utilizes a biosignature of OTUs. As used herein, the term "biosignature" refers to an association of the level of one or more members of one or more OTUs with a particular condition, such as a classification, diagnosis, prognosis, and/or predicted outcome of a pulmonary condition in a subject. In one embodiment, the biosignature comprises a determination of the presence, absence, and/or quantity of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 250, 500, 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 250,000, 500,000 or 1,000,000 OTUs in a sample using a single assay. In some embodiments, the biosignature comprises the presence of or changes in the level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, or more OTUs. In some embodiments, OTUs in a biosignature comprise OTUs selected from one or more of Table 3, Table 4, or Table 5.

[0069] In one embodiment, the biosignature is associated with a single condition, for example a single pulmonary condition. In another embodiment, the biosignature is associated with a combination of conditions, for example two or more pulmonary conditions, or one or more pulmonary conditions combined with one or more non-pulmonary conditions. In some embodiments, the condition is chronic obstructive pulmonary disease. A biosignature can be obtained for any sample, including but not limited to: tissue samples; cell culture samples; bacterial culture samples; samples obtained from a subject, including biopsies, body fluids and other excreted material; pulmonary samples; other samples as described herein; materials derived therefrom; and combinations thereof. In some embodiments, the sample is a pulmonary sample. In some embodiments, the pulmonary sample is sputum, endotracheal aspirate, bronchoalveolar lavage sample, a swab of the endotrachea, materials derived therefrom, or combinations thereof. In some embodiments, a biosignature of a test sample is compared to a known biosignature, and a determination is made as to likelihood that the biosignatures are the same. In some embodiments, a biosignature of a sample is compared to a biosignature for a classification, diagnosis, prognosis, and/or predicted outcome of a pulmonary condition. The biosignature to which the biosignature of the test sample is compared can be determined before, after, or at substantially the same time as that of the test sample. Biosignatures can be the result of one or more analyses of one or more samples from a particular source. In some embodiments, a biosignature is indicative of a response to treatment. In some embodiments, a biosignature is used as a basis for the selection of a mode of treatment.

[0070] In some embodiments, the biosignature of a test sample is a combination of two or more independent biosignatures, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independent biosignatures. In one embodiment, each of the two or more biosignatures contained in a sample are assayed simultaneously. In a further embodiment, a subset of biosignatures can be evaluated through the use of low-density detection systems, comprising the determination of the presence, absence, and/or level of no more than 10, 25, 50, 100, 250, 500, 1000, 2000, or 5000 OTUs.

[0071] In some embodiments, a biosignature comprises a measure of the number of members in one or more bacterial families or OTUs. The number of members may range from 0 to 10000 or more, such as 0 to 5000, 0 to 2500, 0 to 1000, 0 to 2000, 0 to 1000, 0 to 900, 0 to 800, 0 to 700, 0 to 600, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 50, 0 to 25, 0 to 20, 0 to 10, or 0 to 5. In some embodiments, a biosignature comprises the presence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 2500, 5000, 10000, or more members of one or more bacterial families or OTUs, or the presence of a range that includes any two of these values as end points. In some embodiments, a biosignature comprises a ratio between numbers of members in two or more bacterial families or OTUs. The numerator and denominator of such ratios may include overlapping sets of bacterial families or OTUs. Ratios of the numbers of members in two or more bacterial families may compare a first set of one or more bacterial families or OTUs to a second set of one or more bacterial families or OTUs, where there is at least one bacterial family or OTU difference between the first and second set. A set of bacterial families or OTUs may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more OTUs. Bacterial families or OTUs may be selected from one or more of Table 3, Table 4, or Table 5. Examples of bacterial families or OTUs include, but are not limited to, Campylobacteraceae, Porphyromonadaceae, Prevotellaceae, Corynebacteriaceae, Enterobacteriaceae, Alteromonadaceae,

Peptococc/Acidaminococcacea, Lactobacillaceae, Enterococcaceae, Pasteurellaceae, Flavobacteriaceae, Acidobacteriaceae, Staphylococcaceae, Micrococcaceae, Peptostreptococcaceae, Helicobacteraceae, Streptococcaceae, Pseudomonadaceae, Bacillaceae, Clostridiaceae, Mollicutes, Cyanobacteria, Anaerolineae, Sphingobacteria, Acidobacteria, Flavobacteria, Alphaproteobacteria, Bacteroidetes, Epsilonproteobacteria, Betaproteobacteria, Deltaproteobacteria, Actinobacteria, Gammaproteobacteria, Bacilli, Clostridia, Moraxellaceae, Chloroplasts, Peptostreptococcaceae, Spirochaetaceae, Lachnospiraceae, Verrucomicrobiae, Corynebacteriaceae, Bifidobacteriaceae, Micromonosporaceae, Desulfotomaculum, Dehalococcoidetes, and bacterial families or OTUs as described in the drawings.

[0072] In one aspect, the invention provides methods, systems, and compositions for detecting and identifying a plurality of biomolecules and/or organisms in a sample. The invention utilizes the ability to differentiate between individual organisms or OTUs. In one aspect, the individual organisms or OTUs are identified using organism-specific and/or OTU-specific probes, e.g., oligonucleotide probes. More specifically, some embodiments relate to selecting organism-specific and/or OTU-specific oligonucleotide probes useful in detecting and identifying biomolecules and organisms in a sample. In some embodiments, an oligonucleotide probe is selected on the basis of the cross-hybridization pattern of the oligonucleotide probe to regions within a target oligonucleotide and its homologs in a plurality of organisms. The homologs can have nucleotide sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical. Such oligonucleotides can be gene, or intergenetic sequences, in whole or a portion thereof. The oligonucleotides can range from 10 to over 10,000 nucleotides in length. In some other embodiments, a method is provided for detecting the presence of an OTU in a sample based at least partly on the cross-hybridization of the OTU-specific oligonucleotide probes to probes specific for other organisms or OTUs. In some embodiments, the biosignature to which a sample biosignature is compared comprises a positive result for the presence of the targets for one or more probes.

[0073] In one aspect, the invention provides a diagnostic system for the determination or evaluation of a biosignature of a sample. In one embodiment, the diagnostic system comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, or more probes. In another embodiment, the diagnostic system comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, or more probes.

High Capacity Systems

[0074] In one aspect of the invention, a high capacity system is provided for determining a biosignature of a sample by assessing the total microorganism population of a sample in terms of the microorganisms present and optionally their percent composition of the total population. In some embodiments, the system comprises a plurality of probes that are capable of determining the presence or quantity of at least 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, or more different OTUs in a single assay. In some embodiments, one or more OTUs are selected from one or more of Table 3, Table 4, or Table 5. Typically, the probes selectively hybridize to a highly conserved polynucleotide. Usually, the probes hybridize to the same highly conserved polynucleotide or within a portion thereof. Generally, the highly conserved polynucleotide or fragment thereof comprises a gene or fragment thereof. Non-limiting examples of highly conserved polynucleotides comprise nucleotide sequences found in the 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene and nifD gene. In other embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 15 or more, 20 or more, 25 or more, or 50 or more collections of probes are employed, each of which specifically hybridizes to a different highly conserved polynucleotide or portion thereof. For example, a first collection of probes binds to the same region of the 16S rRNA gene, a second collection of probes binds to the same region of the 16S rRNA gene that is different from the region bound by probes in the first collection, and a third collection of probes binds to the same region of the 23 S rRNA gene. The use of two or more collections of probes where each collection recognizes distinct and separate highly conserved polynucleotides or portions thereof allows for the generation and testing of more probes the use of which can provide greater discrimination between species or OTUs.

[0075] Highly conserved polynucleotides usually show at least 80%, 85%, 90%, 92%, 94%, 95%, or 97% homology across a domain, kingdom, phylum, class, order, family or genus, respectively. The sequences of these polynucleotides can be used for determining evolutionary lineage or making a phylogenetic determination and are also known as phylogenetic markers. In some embodiments, a biosignature comprises the presence, absence, and/or abundance of a combination of phylogenetice markers. The OTUs detected by the probes disclosed herein can be bacterial, archeal, fungal, or eukaryotic in origin. Additionally, the methodologies disclosed herein can be used to quantify OTUs that are bacterial, archaeal, fungal, or eukaryotic. By combining the various probe sets, a system for the detection of bacteria, archaea, fungi, eukaryotes, or combinations thereof can be designed. Such a universal microorganism test that is conducted as a single assay can provide great benefit for assessing and understanding the composition and ecology of numerous environments, including characterization of biosignatures for various samples, environments, conditions, and contaminants.

[0076] In another aspect of the invention, a system is provided that is capable of determining the probability of presence and optionally quantity of at least 10,000, 20,000, 30,000, 40,000, 50,000 or 60,000 different OTUs of a single domain in a single assay. Such a system makes a probability determination with a confidence level greater than 90%, 91%, 92%, 93%, 94%, 95%, 99% or 99.5%. In some embodiments, a biosignature can comprise the combined result of each probability determination.

[0077] Some embodiments provide a method of selecting an oligonucleotide probe that is specific for a node in a clustering tree. In some embodiments, the method comprises selecting a highly conserved target polynucleotide and its homologs for a plurality of organisms; clustering the polynucleotides and homologs of the plurality of organisms into a clustering tree; and determining a cross-hybridization pattern of a candidate oligonucleotide probe that hybridizes to a first polynucleotide to each node on the clustering tree. This determination is performed (e.g., in silico) to determine the likelihood that the probe would cross hybridize with homologs of its target complementary sequence. The candidate oligonucleotide probe can be complementary to a highly conserved target polynucleotide, a fragment of the highly conserved target or one of its homologs in one of the plurality of organisms. In some embodiments, a method is provided for the determination of the cross-hybridization pattern of a variant of the candidate oligonucleotide probe to each node on the clustering tree, wherein the variant corresponds to the candidate oligonucleotide probe but comprises at least 1 nucleotide mismatch; and selecting or rejecting the candidate oligonucleotide probe on the basis of the cross-hybridization pattern of the candidate oligonucleotide probe and the cross-hybridization pattern of the variant. In some embodiments, the node is an operational taxon unit (OTU). In some embodiments, the node is a single organism.

[0078] Some embodiments provide a method of selecting an OTU-specific oligonucleotide probe for use in detecting a plurality of organisms in a sample. In some embodiments, the method comprises: selecting a highly conserved target polynucleotide and its homologs from the plurality of organisms; clustering the polynucleotides of the target gene and its homologs from the plurality of organisms into one or more operational taxonomic units (OTUs), wherein each OTU comprises one or more groups of similar nucleotide sequence; determining the cross-hybridization pattern of a candidate OTU-specific oligonucleotide probe to the OTUs, wherein the candidate OTU-specific oligonucleotide probe corresponds to a fragment of the target gene or its homolog from one of the plurality of organisms; determining the cross-hybridization pattern of a variant of the candidate OTU-specific oligonucleotide probe to the OTUs, wherein the variant comprises at least 1 nucleotide mismatch from the candidate OTU-specific oligonucleotide probe; and selecting or rejecting the candidate OTU-specific oligonucleotide probe on the basis of the cross-hybridization pattern of the candidate OTU-specific oligonucleotide probe and the cross-hybridization pattern of the variant. In some embodiments, the candidate OTU-specific oligonucleotide probe is selected if the candidate OTU-specific oligonucleotide probe does not cross-hybridize with any polynucleotide that is complementary to probes from other OTUs. In further embodiments, the candidate OTU-specific oligonucleotide probe is selected if the candidate OTU-specific oligonucleotide probe cross-hybridizes with the polynucleotide in no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 100, 200, 500, or 1000 other OTU groups.

[0079] Some embodiments provide a method of selecting a set of organism-specific oligonucleotide probes for use in detecting a plurality of organisms in a sample. In some embodiments, the method comprises: identifying a highly conserved target polynucleotide and its homologs in the plurality of organisms; determining the cross-hybridization pattern of a candidate organism-specific oligonucleotide probe to the sequences of the highly conserved target polynucleotide and its homologs in the plurality of organisms, wherein the candidate oligonucleotide probe corresponds to a fragment of the target sequence or its homolog from one of the plurality of organisms; determining the cross-hybridization pattern of a variant of the candidate organism-specific oligonucleotide probe to the sequences of the highly conserved target sequence and its homologs in the plurality of organisms, wherein the variant comprises at least 1 nucleotide mismatch from the candidate organism-specific oligonucleotide probe; and selecting or rejecting the candidate organism-specific oligonucleotide probe on the basis of the cross-hybridization pattern of the candidate organism-specific oligonucleotide probe and the cross-hybridization pattern of the variant of the candidate organism-specific oligonucleotide probe.

[0080] In some embodiments, an OTU-specific oligonucleotide probe does not cross-hybridize with any polynucleotide that is complementary to probes from other OTUs. In other embodiments, an OTU- specific oligonucleotide probe cross-hybridizes with the polynucleotide in no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 30, 40, 50, 100, 200, 500, or 1000 other OTU groups. Some embodiments utilize a set of organism-specific oligonucleotide probes for use in detecting a plurality of organisms in a sample. In further embodiments, the candidate organism-specific oligonucleotide probe is selected if the candidate organism-specific oligonucleotide probe only hybridizes with the target nucleic acid molecule of no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50 unique organisms in the plurality of organisms. In other embodiments, the process is iterative with multiple candidate specific- specific oligonucleotide probes selected. Frequently, the selected organism-specific oligonucleotide probes are clustered and aligned into groups of similar sequences that allow for the detection of an organism with high confidence based on no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, or 60 organism-specific oligonucleotide probe matches per OTU. Generally, the candidate organism that the organism-specific oligonucleotide probes detect corresponds to a leaf or node of at least one phylogenetic, genealogic, evolutionary, or taxonomic tree. Knowledge of the position that a candidate organism detected by the organism-specific oligonucleotide probe occupies on a tree provides relational information of the organism to other members of its domain, phylum, class, subclass, order, family, subfamily, or genus. [0081] In some embodiments, the method disclosed herein selects and/or utilizes a set of organism- specific oligonucleotide probes that are a hierarchical set of oligonucleotide probes that can be used to detect and differentiate a plurality of organisms. In some embodiments, the method selects and/or utilizes organism-specific or OTU-specific oligonucleotide probes that allow a comprehensive screen for at least 80%, 85%, 90%, 95%, 99% or 100% of all known bacterial or archaeal taxa in a single analysis, and thus provides an enhanced detection of different desired taxonomic groups. In some embodiments, the identity of all known bacterial or archaeal taxa comprises taxa that were previously identified by the use of oligonucleotide specific probes, PCR cloning, and sequencing methods. Some embodiments provide methods of selecting and/or utilizing a set of oligonucleotide probes capable of correctly categorizing mixed target nucleic acid molecules into their proper operational taxonomic unit (OTU) designations. Such methods can provide comprehensive prokaryotic or eukaryotic identification, and thus comprehensive biosignature characterization.

[0082] In some embodiments, the selected OTU-specific oligonucleotide probe is used to calculate the relative abundance of one or more organisms that belong to a specific OTU at differing levels of taxonomic identification. In some embodiments, an array or collection of microparticles comprising at least one organism-specific or OTU-specific oligonucleotide probe selected by the method disclosed herein is provided to infer specific microbial community activities. For example, the identity of individual taxa in a microbial consortium from an anaerobic environment for instance, a marsh, can be determined along with their relative abundance. If the consortium is suspected of harboring microorganisms capable of butanol fermentation, then after providing a suitable feedstock in an anaerobic environment if the production of butanol is noted, then those taxa responsible for butanol fermentation can be inferred by the microorganisms that have abundant quantities of 16S rRNA. The invention provides methods to measure taxa abundance based on the detection of directly labeled 16S rRNA.

[0083] In some embodiments, multiple probes are selected for increasing the confidence level and/or sensitivity level of identification of a particular organism or OTU. The use of multiple probes can greatly increase the confidence level of a match to a particular organism. In some embodiments, the selected organism-specific oligonucleotide probes are clustered and aligned into groups of similar sequence such that detection of an organism is based on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 or more oligonucleotide probe matches. In some embodiments, the oligonucleotide probes are specific for a species. In other embodiments, the oligonucleotide probe recognizes related organisms such as organisms in the same subgenus, genus, subfamily, family, suborder, order, sub-class, class, sub-phylum, phylum, sub-kingdom, or kingdom.

[0084] Perfect match (PM) probes are perfectly complementary to the target polynucleotide, e.g., a sequence that identifies a particular organism. In some embodiments, a system of the invention comprises mismatch (MM) control probes. Usually, MM probes are otherwise identical to PM probes, but differ by one or more nucleotides. Probes with one or more mismatch can be used to indicate non- specific binding and a possible non-match to the target sequence. In some embodiments, the MM probes have one mismatch located in the center of the probe, e.g., in position 13 for a 25mer probe. The MM probe is scored in relation to its corresponding PM probe as a "probe pair." MM probes can be used to estimate the background hybridization, thereby reducing the occurrence of false positive results due to non-specific hybridization, a significant problem with many current detection systems. If an array is used, such as an Affymetrix high density probe array or Illumina bead array, ideally, the MM probe is positioned adjacent or close to its corresponding PM probe on the array.

[0085] Some embodiments relate to a method of selecting and/or utilizing a set of oligonucleotide probes that enable simultaneous identification of multiple prokaryotic taxa with a relatively high confidence level. Typically, the confidence level of identification is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%. In general, an OTU refers to an individual species or group of highly related species that share an average of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 99.5%, or more sequence homology in a highly conserved region. Multiple MM probes may be utilized to enhance the quantification and confidence of the measure. In some embodiments, each interrogation probe of a plurality of interrogation probes has from about 1 to about 20 corresponding mismatch control probes. In further embodiments, each interrogation probe has from about 1 to about 10, about 1 to about 5, about 1 to 4, 1 to 3, 2 or 1 corresponding mismatch probes. These interrogation probes target unique regions within a target nucleic acid sequence, e.g., a 16S rRNA gene, and provide the means for identifying at least about 10, 20, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 250,000, 500,000 or 1,000,000 taxa. In some embodiments, multiple targets can be simultaneously assayed or detected in a single assay through a high- density oligonucleotide probe system. The sum of all target hybridizations is used to identify specific prokaryotic taxa. The result is a more efficient and less time consuming method of identifying unculturable or unknown organisms. The invention can also provide results that could not previously be achieved, e.g., providing results in hours where other methods would require days. In some embodiments, a microbiome (i.e., sample) can be assayed to determine the identity and optionally the abundance of its constituent microorganisms in less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour.

[0086] In some embodiments, the set of OTU-specific oligonucleotide probes comprises from about 1 to about 500 probes for each taxonomic group. In some embodiments, the probes are proteins including antibodies, or nucleic acid molecules including oligonucleotides or fragments thereof. In some embodiments, an oligonucleotide probe corresponds to a nucleotide fragment of the target nucleic acid molecule. In some embodiments, from about 1 to about 500, about 2 to about 200, about 5 to about 150, about 8 to about 100, about 10 to about 35, or about 12 to about 30 oligonucleotide probes can be designed for each taxonomic grouping. In other embodiments, a taxonomic group can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, or more probes. In some embodiments, various taxonomic groups can have different numbers of probes, while in other embodiments, all taxonomic groups have a fixed number of probes per group. Multiple probes in a taxonomic group can provide additional data that can be used to make a determination, also known as "making a call" as to whether an OTU is present or not. Multiple probes also allow for the removal of one or more probes from the analysis based on insufficient signal strength, cross hybridization or other anomalies. Removing probes can increase the confidence level of results and further allow for the detection of low abundant microorganisms. The oligonucleotide probes can each be from about 5 to about 100 nucleotides, from about 10 to about 50 nucleotides, from about 15 to about 35 nucleotides, or from about 20 to about 30 nucleotides. In some embodiments, the probes are at least 5- mers, 6-mers, 7-mers, 8-mers, 9-mers, 10-mers, 11-mers, 12-mers, 13-mers, 14-mers, 15-mers, 16-mers, 17-mers, 18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, 25-mers, 26-mers, 27-mers, 28-mers, 29-mers, 30-mers, 31-mers, 32-mers, 33-mers, 34-mers, 35-mers, 36-mers, 37-mers, 38-mers, 39-mers, 40-mers, 41-mers, 42-mers, 43-mers, 44-mers, 45-mers, 46-mers, 47-mers, 48-mers, 49-mers, 50-mers, 51-mers, 52-mers, 53-mers, 54-mers, 55-mers, 56-mers, 57-mers, 58-mers, 59-mers, 60-mers, 61-mers, 62-mers, 63-mers, 64-mers, 65-mers, 66-mers, 67-mers, 68-mers, 69-mers, 70-mers, 71-mers, 72-mers, 73-mers, 74-mers, 75-mers, 76-mers, 77-mers, 78-mers, 79-mers, 80-mers, 81-mers, 82-mers, 83-mers, 84-mers, 85-mers, 86-mers, 87-mers, 88-mers, 89-mers, 90-mers, 91-mers, 92-mers, 93-mers, 94-mers, 95-mers, 96-mers, 97-mers, 98-mers, 99-mers, 100-mers or combinations thereof

[0087] Some embodiments provide methods of selecting multiple, confirmatory, organism-specific or OTU-specific probes to increase the confidence of detection. In some embodiments, the methods also select one or more mismatch (MM) probes for every perfect match (PM) probe to minimize the effect of cross-hybridization by non-target regions. The organism-specific and OTU-specific oligonucleotide probes selected by the methods disclosed herein can simultaneously identify thousands of taxa present in an environmental sample and allow accurate identification of microorganisms and their phylogenetic relationships in a community of interest. Systems that use the organism-specific and OTU-specific oligonucleotide probes selected by the methods disclosed herein and the computational analysis disclosed herein have numerous advantages over rRNA gene sequencing techniques. Such advantages include reduced cost per microbiome analysis, and increased processing speed per sample or microbiome from both the physical analysis and the computational analysis point of view. In general, the analysis procedures are not adversely affected by chimeras, are not subject to creating artificial phylotypes, and are not subject to barcode PCR bias. Additionally, quantitative standards can be run with a microbiome sample of the invention.

[0088] Some embodiments provide a method for selecting and/or utilizing a set of OTU- or organism- specific oligonucleotide probes for use in an analysis system or bead multiplex system for simultaneously detecting a plurality of organisms in a sample. The method targets known diversity within target nucleic acid molecules to determine microbial community composition and establish a biosignature. The target nucleic acid molecule is typically a highly conserved polynucleotide. In some embodiments, the highly conserved polynucleotide is from a highly conserved gene, whereas in other embodiments the polynucleotide is from a highly conserved region of a gene with moderate or large sequence variation. In further embodiments, the highly conserved region may be an intron, exon, or a linking section of nucleic acid that separates two genes. In some embodiments, the highly conserved polynucleotide is from a "phylogenetic" gene. Phylogenetic genes include, but are not limited to, the 5.8S rRNA gene, 12S rRNA gene, 16S rRNA gene-prokaryotic, 16S rRNA gene-mitochondrial, 18S rRNA gene, 23 S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, and the nifD gene. With eukaryotes, the rRNA gene can be nuclear, mitochondrial, or both. In some embodiments, the 16S-23S rRNA gene internal transcribed spacer (ITS) can be used for differentiation of closely related taxa with or without the use of other rRNA genes. For example, rRNA, e.g., 16S or 23 S rRNA, acts directly in the protein assembly machinery as a functional molecule rather than having its genetic code translated into protein. Due to structural constraints of 16S rRNA, specific regions throughout the gene have a highly conserved polynucleotide sequence; although, non-structural segments may have a high degree of variability. Probing the regions of high variability can be used to identify OTUs that represent a single species level, while regions of less variability can be used to identify OTUs that represent a subgenus, a genus, a subfamily, a family, a sub-order, an order, a sub-class, a class, a sub-phylum, a phylum, a sub- kingdom, or a kingdom. The methods disclosed herein can be used to select organism-specific and OTU- specific oligonucleotide probes that offer a high level of specificity for the identification of specific organisms, OTUs representing specific organisms, or OTUs representing specific taxonomic group of organisms. The systems and methods disclosed herein are particularly useful in identifying closely related microorganisms and OTUs from a background or pool of closely related organisms.

[0089] The probes selected and/or utilized by the methodologies of the invention can be organized into OTUs that provide an assay with a sensitivity and/or specificity of more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, sensitivity and specificity depends on the hybridization signal strength, number of probes in the OTU, the number of potential cross hybridization reactions, the signal strength of the mismatch probes, if present, background noise, or combinations thereof. In some embodiments, an OTU containing one probe may provide an assay with a sensitivity and specificity of at least 90%, while another OTU may require at least 20 probes to provide an assay with sensitivity and specificity of at least 90%.

[0090] Some embodiments relate to methods for phylogenetic analysis system design and signal processing and interpretation for use in detecting and identifying a plurality of biomolecules and organisms in a sample. More specifically, some embodiments relate to a method of selecting a set of organism-specific oligonucleotide probes for use in detecting a plurality of organisms in a sample with a high confidence level. Some embodiments relate to a method of selecting a set of OTU-specific oligonucleotide probes for use in detecting a plurality of organisms in a sample with a high confidence level.

[0091] In the case of highly conserved polynucleotides like 16S rR A that may have only one to a few nucleotides of sequence variability over any 15- to 30-bp region targeted by probes for discrimination between related microbial species, it is advantageous to maximize the probe-target sequence specificity in an assay system. Some embodiments of the present invention provide methods of selecting organism- specific oligonucleotide probes that effectively minimize the influence of cross-hybridization. In one embodiment, the method comprises: (a) identifying sequences of a target nucleic acid molecule corresponding to the plurality of organisms; (b) determining the cross-hybridization pattern of a candidate organism-specific oligonucleotide probe to the target nucleic acid molecule from the plurality of organisms, wherein the candidate oligonucleotide probe corresponds to a sequence fragment of the target nucleic acid molecule from the plurality of organisms; (c) determining the cross-hybridization pattern of a variant of the candidate organism-specific oligonucleotide probe to the target nucleic acid molecule from the plurality of organisms, wherein the variant of the candidate organism-specific oligonucleotide probe comprises at least 1 nucleotide mismatch compared to the candidate organism-specific oligonucleotide probe ; and (d) selecting or rejecting the candidate organism-specific oligonucleotide probe on the basis of the cross-hybridization pattern of the candidate organism-specific oligonucleotide probe and the cross- hybridization pattern of the variant of the candidate organism-specific oligonucleotide probe. In some embodiments, a method of selecting a set of OTU-specific oligonucleotide probes for use in detecting a plurality of organisms in a sample is provided. In some embodiments, the method comprises: (a) identifying sequences of a target nucleic acid molecule corresponding to the plurality of organisms; (b) clustering the sequences of the target nucleic acid molecule from the plurality of organisms into one or more Operational Taxonomic Units (OTUs), wherein each OTU comprises one or more groups of similar sequences; (c) determining the cross-hybridization pattern of a candidate OTU-specific oligonucleotide probe to the OTUs, wherein the candidate OTU-specific oligonucleotide probe corresponds to a sequence fragment of the target nucleic acid molecule from one of the plurality of organisms; (d) determining the cross-hybridization pattern of a variant of the candidate OTU-specific oligonucleotide probe to the OTUs, wherein the variant of the candidate OTU-specific oligonucleotide probe comprises at least 1 nucleotide mismatch compared to the candidate OTU-specific oligonucleotide probe ; and (e) selecting or rejecting the candidate OTU-specific oligonucleotide probe on the basis of the cross-hybridization pattern of the candidate OTU-specific oligonucleotide probe to the OTUs and the cross-hybridization pattern of the variant of the candidate OTU-specific oligonucleotide probe to the OTUs. In some embodiments, candidate OTU-specific oligonucleotide probe are rejected when the candidate OTU-specific oligonucleotide probe or its variant are predicted to cross-hybridize with other target sequences. In some embodiments, a predetermined amount of predicted cross-hybridization is allowed. [0092] In some embodiments, selected oligonucleotide probes are synthesized by any relevant method known in the art. Some examples of suitable methods include printing with fine-pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry. In one example, a photolithographic method can be used to directly synthesize the chosen oligonucleotide probes onto a surface. Suitable examples for the surface include glass, plastic, silicon and any other surface available in the art. In certain examples, the oligonucleotide probes can be synthesized on a glass surface at an approximate density from about 1,000 probes per μπι ² to about 100,000 probes per μπι ² , preferably from about 2000 probes per μηΑο about 50,000 probes per μπι ² , more preferably from about 5000 probes per μπΑο about 20,000 probes per μπι ² . In one example, the density of the probes is about 10,000 probes per μπι ² . The number of probes on the array can be quite large e.g., at least 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ^s or 10 ⁹ probes per array. Usually, for large arrays only a relatively small proportion (i.e., less than about 1%, 0.1% 0.01%, 0.001%, 0.00001%, 0.000001% or 0.0000001%) of the total number of probes of a given length target an individual OTU. Frequently, lower limit arrays have no more than 10, 25, 50, 100, 500, 1,000, 5,000, or 10,000, 25,000, 50,000, 100,000 or 250,000 probes.

[0093] Typically, the arrays or microparticles have probes to one or more highly conserved polynucleotides. The arrays or microparticles may have further probes (e.g. confirmatory probes) that hybridize to functionally expressed genes, thereby providing an alternate or confirmatory signal upon which to base the identification of a taxon. For example, an array may contain probes to 16S rR A gene sequences from Yersinia pestis and Vibrio cholerae and also confirmatory probes to Y. pestis cafl virulence gene or V. cholerae zonula occludens toxin (zot) gene. The detection of hybridization signals based on probes binding to 16S rRNA polynucleotides associated with a particular OTU coupled with the detection of a hybridization signal based on a confirmatory probe can provide a higher level of confidence that the OTU is present. For instance, if hybridization signals are detected for the probes associated Y. pestis OTU and the confirmatory probe also displays a hybridization signal for the expression of Y. pestis cafl then the confidence level subscribed to the presence or quantity of Y. pestis will be higher than the confidence level obtained from the use of OTU probes alone.

[0094] A range of lengths of probes can be employed on the arrays or microparticles. As noted above, a probe may consist exclusively of a complementary segments, or may have one or more complementary segments juxtaposed by flanking, trailing and/or intervening segments. In the latter situation, the total length of complementary segment(s) can be more important that the length of the probe. In functional terms, the complementary segment(s) of the PM probes should be sufficiently long to allow the PM probes to hybridize more strongly to a target polynucleotide e.g., 16S rRNA, compared with a MM probe. A PM probe usually has a single complementary segment having a length of at least 15 nucleotides, and more usually at least 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or 30 bases exhibiting perfect complementarity. [0095] In some arrays or lots of microparticles, all probes are the same length. In other arrays or lots of microparticles, probe length varies between quantification standard (QS) probes, negative control (NC) probes, probe pairs, probe sets (OTUs) and combinations thereof. For example, some arrays may have groups of OTUs that comprise probe pairs that are all 23 mers, together with other groups of OTUs or probe sets that comprise probe pairs that are all 25 mers. Additional groups of probes pairs of other lengths can be added. Thus, some arrays may contain probe pairs having sizes of 15 mers, 16mers, 17mers, 18mers, 19mers, 20mers, 21mers, 22mers, 23mers, 24mers, 25 mers, 26mers, 27 mers, 28mers, 29 mers, 30mers, 31mers, 32mers, 33mers, 34mers, 35mers, 36mers, 37mers, 38mers, 39mers, 40mers or combinations thereof. Other arrays may have different size probes within the same group, OTU, or probe set. In these arrays, the probes in a given OTU or probe set can vary in length independently of each other. Having different length probes can be used to equalize hybridization signals from probes depending on the hybridization stability of the oligonucleotide probe at the pH, temperature, and ionic conditions of the reaction.

[0096] In another aspect of the invention, a system is provided for determining the presence or quantity of a plurality of different OTUs in a single assay where the system comprises a plurality of polynucleotide interrogation probes, a plurality of polynucleotide positive control probes, and a plurality of polynucleotide negative control probes. In some embodiments, the system is capable of detecting the presence, absence, relative abundance, and/or quantity of at least 5, 10, 20, 50, 100, 250, 500, 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 250,000, 500,000 or 1 ,000,000 OTUs in a sample using a single assay. In some embodiments, the polynucleotide positive control probes include 1) probes that target sequences of prokaryotic or eukaryotic metabolic genes spiked into the target nucleic acid sequences in defined quantities prior to fragmentation, or 2) probes complimentary to a pre- labeled oligonucleotide added into the hybridization mix after fragmentation and labeling. The control added prior to fragmentation collectively tests the fragmentation, biotinylation, hybridization, staining and scanning efficiency of the system. It also allows the overall fluorescent intensity to be normalized across multiple analysis components used in a single or combined experiment, such as when two or more arrays are used in a single experiment or when data from two separate experiments is combined. The second control directly assays the hybridization, staining and scanning of the system. Both types of control can be used in a single experiment.

[0097] In some embodiments, the QS standards (positive controls) are PM probes. In other embodiments, the QS standards are PM and MM probe pairs. In further embodiments, the QS standards comprise a combination of PM and MM probe pairs and PM probes without corresponding MM probes. In another embodiment, the QS standards comprise at least one, two, three, four, five, six, seven, eight, nine, ten or more MM probes for each corresponding PM probe. In a further embodiment, the QS standards comprise at least one, two, three, four, five, six, seven, eight, nine, ten or more PM probes for each corresponding MM probe. A system can comprise at least 1 positive control probe for each 1, 10, 100, or 1000 different interrogation probes.

[0098] In some cases, the spiked-in oligonucleotides that are complementary to the positive control probes vary in G+C content, uracil content, concentration, or combinations thereof. In some embodiments, the G+C% ranges from about 30% to about 70%, about 35% to about 65% or about 40% to about 60%. QS standards can also be chosen based on the uracil incorporation frequency. The QS standards may incorporate uracil in a range from about 1 in 100 to about 60 in 100, about 4 in 100 to about 50 in 100, or about 10 in 100 to about 50 in 100. In some cases, the concentration of these added oligonucleotides will range over 1, 2, 3, 4, 5, 6, or 7 orders of magnitude. Concentration ranges of about 10 ⁵ to 10 ¹⁴ , 10 ⁶ to 10 ¹³ , 10 ⁷ to 10 ¹² , 10 ⁷ to 10 ¹¹ , 10 ⁸ to 10 ^u , and 10 ⁸ to 10 ¹⁰ can be employed and generally feature a linear hybridization signal response across the range. In some embodiments, positive control probes for the conduction of the methods disclosed herein comprise polynucleotides that are complementary to the positive control sequences shown in Table 6. Other genes that can be used as targets for positive controls include genes encoding structural proteins, proteins that control growth, cell cycle or reproductive regulation, and house keeping genes. Additionally, synthetic genes based on highly conserved genes or other highly conserved polynucleotides can be added to the sample. Useful highly conserved genes from which synthetic genes can be designed include 16S rRNA genes, 18S rRNA genes, 23SrRNA genes. Exemplary control probes are provided as SEQ ID NOs:51-100.

Table 6 Positive Control Sequences

Synthetic 16S rRNA

controls

SYNM neurolyt_st Synthetic derivative of Mycoplasma neurolyticum 16S rRNA gene

SYNLc.oenos_st Synthetic derivative of Leuconostoc oenos 16S rRNA gene

SYNCau.cres8_st Synthetic derivative of Caulobacter crescenins 16S rRNA gene

SYNFer.nodosm st Synthetic derivative of Fervidobacterium nodosum 16S rRNA gene

SY Sap.grandi_st Synthetic derivative of Saprospira grandis 16S rRNA gene

[0099] In some embodiments, the negative controls comprise PM and MM probe pairs. In further embodiments, the negative controls comprise a combination of PM and MM probe pairs and PM probes without corresponding MM probes. In other embodiments, the negative control probes comprise at least one, two, three, four, five, six, seven, eight, nine, ten or more MM probes for each corresponding negative control PM probe. A system can comprise at least 1 negative control probe for each 1, 10, 100, or 1000 different interrogation probes (PMs).

[00100] Generally, the negative control probes hybridize weakly, if at all, to 16S rRNA gene or other highly conserved gene targets. The negative control probes can be complementary to metabolic genes of prokaryotic or eukaryotic origin. Generally, with negative control probes, no target material is spiked into the sample. In some embodiments, negative control probes are from the same collection of probes that are also used for positive controls, but no material complementary to the negative control probes are spiked into the sample, in contrast to the positive control probe methodology. In essence, the control probes are universal control probes and play the role of a positive or negative control probes depending on the system's design. One of skill in the art will appreciate that the universal control probes are not limited to highly conserved sequence analysis systems and have applications beyond the present embodiments disclosed herein.

[00101] In a further embodiment, probes to non-highly conserved polynucleotides are added to a system to provide species-specific identification or confirmation of results achieved with the probes to the highly conserved polynucleotides. Usually, these "confirmatory" probes cross hybridize very weakly, if at all, to highly conserved polynucleotides recognized by the perfect match probes. Useful species-specific genes include metabolic genes, genes encoding structural proteins, proteins that control growth, cell cycle or reproductive regulation, housekeeping genes or genes that encode virulence, toxins, or other pathogenic factors. In some embodiments, the system comprises at least 1, 5, 10, 20, 30, 40, 50 60, 70, 80, 90 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 5,000 or 10,000 species- specific probes.

[00102] In some embodiments, a system of the invention comprises an array. Non-limiting examples of arrays include microarrays, bead arrays, through-hole arrays, well arrays, and other arrays known in the art suitable for use in hybridizing probes to targets. Arrays can be arranged in any appropriate configuration, such as, for example, a grid of rows and columns. Some areas of an array comprise the OTU detection probes whereas other areas can be used for image orientation, normalization controls, signal scaling, noise reduction processing, or other analyses. Control probes can be placed in any location in the array, including along the perimeter of the array, diagonally across the array, in alternating sections or randomly. In some embodiments, the control probes on the array comprise probe pairs of PM and MM probes. The number of control probes can vary, but typically the number of control probes on the array range from 1 to about 500,000. In some embodiments, at least 10, 100, 500, 1,000, 5,000, 10,000, 25,000, 50,000, 100,000, 250,000 or 500,000 control probes are present. When control probe pairs are used, the probe pairs will range from 1 to about 250,000 pairs. In some embodiments, at least 5, 50, 250, 500, 2,500, 5,000, 12,500, 25,000, 50,000, 125,000 or 250,000 control probe pairs are present. The arrays can have other components besides the probes, such as linkers attaching the probes to a support. In some embodiments, materials for fabricating the array can be obtained from Affymetrix (Santa Clara, California), GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) or Agilent Technologies (Palo Alto, California.)

[00103] Besides arrays where probes are attached to the array substrate, numerous other technologies may be employed in the disclosed system for the practice of the methods of the invention. In one embodiment, the probes are attached to beads that are then placed on an array as disclosed by Ng et al. (Ng et al. A spatially addressable bead-based biosensor for simple and rapid DNA detection. Biosensors & Bioelectronics, 23:803-810, 2008).

[00104] In another embodiment, probes are attached to beads or microspheres, the hybridization reactions are performed in solution, and then the beads are analyzed by flow cytometry, as exemplified by the Luminex multiplexed assay system. In this analysis system, homogeneous bead subsets, each with beads that are tagged or labeled with a plurality of identical probes, are combined to produce a pooled bead set that is hybridized with a sample and then analyzed in real time with flow cytometry, as disclosed in US Patent 6,524,793. Bead subsets can be distinguished from each other by variations in the tags or labels, e.g., using variability in laser excitable dye content.

[00105] In a further embodiment, probes are attached to cylindrical glass microbeads as exemplified by the Illumina Veracode multiplexed assay system. Here, subsets of microbeads embedded with identical digital holographic elements are used to create unique subsets of probe-labeled microbeads. After hybridization, the microbeads are excited by laser light and the microbead code and probe label are read in real time multiplex assay.

[00106] In another embodiment, a solution based assay system is employed as exemplified by the NanoString nCounter Analysis System (Geiss G et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nature Biotech. 26:317-325, 2008). With this methodology, a sample is mixed with a solution of reporter probes that recognize unique sequences and capture probes that allow the complexes formed between the nucleic acids in the sample and the reporter probes to be immobilized on a solid surface for data collection. Each reporter probe is color-coded and is detected through fluorescence.

[00107] In a further embodiment, branched DNA technology, as exemplified by Panomics QuantiGene Plex 2.0 assay system, is used. Branched DNA technology comprises a sandwich nucleic acid hybridization assay for RNA detection and quantification that amplifies the reporter signal rather than the sequence. By measuring the RNA at the sample source, the assay avoids variations or errors inherent to extraction and amplification of target polynucleotides. The QuantiGene Plex technology can be combined with multiplex bead based assay system such as the Luminex system described above to enable simultaneous quantification of multiple RNA targets directly from whole cells or purified RNA preparations.

Probes and the Selection Thereof

[00108] An exemplary process 300 for the design of target probes for use in the simultaneous detection of a plurality of microorganisms is illustrated in Fig. 3. Briefly, sequences are extracted from a database at a state 301. Typically, the database contains phylogenetic sequences or other highly conserved or homologous sequences. The sequences are analyzed for chimeras at a state 302 that are removed from further consideration. Chimeric sequences result from the union of two or more unrelated sequences, typically from different genes. Optionally, sequences can be further analyzed for structural anomalies, such as propensity for hairpin loop formation, at a state 303 with the identified sequences subsequently removed from further consideration. Next, multiple sequence alignments are performed on the remaining sequences in the dataset at a state 304. The aligned sequences are then checked for laboratory artifacts, such as PCR primer sequences, at a state 305, with identified sequences removed from further consideration. The remaining sequences are clustered at a state 306 and perfect match (PM) probes are selected at a state 307 that have perfect complementarity to sections of the clustered sequences. Optionally, sequence coverage heuristics are performed at a state 308 prior to selecting the mismatch (MM) probes at a state 309 for the corresponding PM probes to create probe pairs. Finally, OTUs represented by probe sets comprising a plurality of probe pairs are assembled at a state 310 to construct a hierarchal taxonomy.

[00109] Generally, a database for extraction of sequences to be used for probe selection is chosen based on the particular conserved gene or highly homologous sequence of interest, the total number of sequences within the database, the length of the overall sequences or the length of highly conserved regions within the sequences listed in the database, and the quality of the sequences therein. Typically, between two databases of equal sequence number but of different sequence length, the database with longer target regions of highly conserved sequence will generally contain a larger total number of possible sequences that can be compared. In some embodiments, the sequences are at least 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200, 1,400, 1,600, 1,800, 2,000, 4,000, 8,000, 16,000 or 24,000 nucleotides long. Generally, databases with larger number of total sequences provide more material to compare. In a further embodiment, the database contains at least 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 100,000, 200,000, 500,000, 1,000,000 or 2,000,000 sequence listings. A gene of particular interest for probe construction is 16S rDNA (16S rR A gene). Other conserved genes include 18S rDNA, 23 S rDNA, gyrA, gyrB gene, groEL, rpoB gene,fusA gene, recA gene, sod A, coxl gene, and nifD gene. In a further embodiment, the spacer region between highly conserved segments of two genes can be used. For example, the spacer region between 16S and 23 S rDNA genes can be used in conjunction with conserved sections of the 16S and 23S rDNA.

[00110] In some embodiments, the detection of a biosignature comprises the use of probes designed to hybridize with known or discovered targets within one or more OTUs. In some embodiments, targets are selected from a collection of known targets, such as in a database. In some embodiments of the invention, a database used for the selection of probes comprises at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or up to 100% of the known sequences of the organisms of interest, e.g., of the bacteria, archaea, fungi, eukaryotes, microorganisms, or prokaryotes of interest. The sequences for each individual organism in the database can include more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 95%) of the genome of the organism, or of the non-redundant regions thereof. In some embodiments, the database includes up to 100% of the genome of the organisms whose sequenced are contained therein, or of the non-redundant sequences thereof. A listing of almost 40,000 aligned 16S rDNA sequences greater than 1250 nucleotides in length can be found on the Greengenes web application, a publicly accessible database run by Lawrence Berkeley National Laboratory. Other publicly accessible databases include GenBank, Michigan State University's ribosomal database project, the Max Planck Institute for Marine Microbiology's Silva database, and the National Institute of Health's NCBI. Proprietary sequence databases or combinations created by amalgamating the contents of two or more private and/or public databases can also be used to practice the methods of this invention. In some embodiments, a sample is assayed for all targets in one or more chosen databases simultaneously. In other embodiments, a sample is assayed for subsets of targets identified in one or more databases simultaneously. In some embodiments, a biosignature comprises the results of assaying a sample for some or all targets in one or more chosen databases. In other embodiments, a biosignature comprises a subset of the results of assaying a sample for some or all targets in one or more chosen databases.

[00111] The analysis of the selected sequences from the database for the detection and removal of chimeras at state 302 is typically performed by generating overlapping fragments and comparing these fragments against each other. Fragments may be retained if they have at least 60%, 70%, 80%, 90%, 95% or 99% sequence identity. It was realized that the above process potentially missed chimeras because the sequence diversity of the selected sequences may be low. By comparing the fragments against a core set of diverse chimera-free sequences, more chimeras can be identified and removed from the sequence set. In cases where one or more sequences are identified that as an ambiguous chimera, e.g., a chimera with a chimeric parent, the chimera is removed and the parent chimera is fragmented and a second comparison cycle is performed. Sequences from a dataset can also be screened for chimeras using a proprietary software program such as Bellerophon3 available from the Greengenes website at greengenes.lbl.gov.

[00112] The dataset of retained non-chimeric sequences can then be screened for structural anomalies at state 303 by aligning the retained sequences against the core set of known sequences. Sequences in the retained dataset that have at least 25, 30, 35, 40, 45, 50, 60, 70 or 80 gaps in their alignment when compared against a core set or have insertions of greater than 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 or 400 basepairs when compared against the core set are tagged as having a sequence anomaly and are removed from the dataset.

[00113] The screened sequences are then aligned into a multiple sequence alignment (MSA) at state 304 for comparison against the known, chimeric free core set. One alignment tool for performing intensive alignment computations is NAST (Nearest Alignment Space Termination) web tool (DeSantis et al., Nucleic Acids Res. (2006) 34:W394-399). Any appropriate alignment tool can be used to compile the MSAs, for example, clustalw (Thompson et al., Nucleic Acids Res (1994) 22:4673^1680) and MUSCLE (Edgar, Nucleic Acids Res. (2004) 32: 1792-1797).

[00114] The aligned sequences are searched for sequences harboring PCR primer sequences at state 305 and any so-identified sequences are removed from the dataset.

[00115] The aligned sequences can then be clustered at the state 306 to create what is termed a "guide tree." First, the sequences are converted to a list of kmers. A pair-wise comparison of the lists of kmers is performed and the percent of kmers in common is recorded in a sparse matrix only if a threshold similarity is found. The sparse matrix is clustered e.g., using complete linkage. Clustering includes agglomerative "bottom-up" or divisive "top-down" hierarchical clustering, distance "partition" clustering and alignment clustering. From each cluster, the sequence with the most information content is chosen as a representative. Usually, sequences derived from genome sequencing projects are given priority in cluster creation because they are less likely to be chimeras or have other sequence anomalies. The cyclic process is repeated using only the representatives from the previous cycle. For each new cycle, the threshold for recording in the sparse matrix is reduced. At the final stage, a root node is linked to the final representative sequences in a multifurcated tree. The representative sequences found in each cycle represent a node in the resulting guide tree. All nodes are linked based on their clustering results via a self-referential table allowing rapid access to any hierarchical point in the guide tree. In some embodiments, the results are stored in a database format, e.g., in a Structured Query Language (SQL) compliant format. In the resulting guide tree, each leaf node represents an individual organism and each node above the lowest level of the guide tree represents a candidate OTU.

[00116] Typical distance matrixes built from approximately 2 x 10 ⁵ sequences can require 40 billion intersections that would require about 40 gigabytes of data space if encoded to disk. Doubling the amount of sequences to 4 x 10 ⁵ requires a quadrupling of the file size (approximately 160GB). The clustering methodology illustrated here using a sparse matrix avoids the need for large files and the expected increase in computing time. Therefore the methodology can be performed more efficiently than conventional sequence clustering methods. Moreover, with distance matrices created from sequence alignments (e.g., DNA alignments), one misalignment can affect many distance values. In contrast, the clustering method illustrated herein is based on the alignment of kmers, and thus the effect of a misalignment on clustering values is significantly reduced.

[00117] Following guide tree construction, the dataset of remaining sequences, now termed the "filtered sequence dataset" is used to select candidate probes, e.g., PM probes. First, unsupported sequence polymorphisms are identified and removed from the filtered sequence dataset using a pre-clustering process that uses the guide tree generated above to create clusters over a minimum similarity and under a maximum size. Typically, clustered sequences are at least 80%, 85%, 90%, 95%, 97% or 99% similar. Usually, clusters have no more than 1,000, 500, 200, 100, 80, 60, 50, 40, 30, 20 or 10 sequences. This process allows sequence data outliers to be detected by comparison within near-neighbors and removed from the filtered sequence dataset.

[00118] Next, the remaining sequences are fragmented to the desired size to generate candidate target probes. Typically, the fragments range from about lOmer to lOOmer, 15mer to about 50mer, about 20mer to about 40mer, about 20mer to about 30mer. Usually, the fragments are at least 15mer, 20mer, 25mer, 30mer, 40mer, 50mer or lOOmer in size. Each candidate target probe is required to be found within a threshold fraction of at least one pre-cluster. Generally, threshold fractions of at least 80%, 90% or 95% are used.

[00119] All candidate PM probes that are within a threshold fraction of at least one pre-cluster are then evaluated for various biophysical parameters, such as melting temperature (61-80° C), G+C content (35- 70%), hairpin energy over -4 kcal/mol, potential for self-dimerization (> 35° C). Candidate PM probes that fall outside of the setting boundaries of the biophysical parameters are eliminated from the dataset. Optionally, probes can be further filtered for ease of photolithographic synthesis.

[00120] The likelihood of cross-hybridization of each PM candidate probe to each non-target input 16s rRNA gene sequence is determined. The cross-hybridization pattern for each PM candidate probe is recorded.

[00121] Sequence coverage heuristics are performed at the state 308 are then applied to candidate PM probes with acceptable biophysical parameters.

[00122] For each candidate PM probe, corresponding MM probes can be generated at the state 309. Each MM probe differs from its corresponding PM probe by at least one nucleotide. In some embodiments, the MM probe differs from its corresponding PM probe by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. Within a MM probe, the mismatched nucleotide or nucleotides can include any of the 3 central bases that are not found in the same position or positions in the PM probe. For example, with a 25mer PM probe that has a guanine at the 13 ^th position, i.e., the central nucleotide, the MM probes comprise probes with adenine, thymine, uracil or cytosine at the 13 position. Similarly, with a 25mer PM probe with an adenine at the 12 ^th nucleotide position and a guanine at the 13 ^th nucleotide position when read from the 3' direction, the possible MM probes comprise probes with guanine at the 12 ^th nucleotide and adenine, thymine or cytosine at the 13 ^th nucleotide position; cytosine at the 12 ^th nucleotide position and adenine, thymine or cytosine at the 13 ^th nucleotide position; and thymine at the 12 ^th nucleotide position and adenine, thymine or cytosine at the 13 ^th nucleotide position. In some embodiments, the mismatched nucleotide or nucleotides include any one or more of the nucleotides in a corresponding PM probe. Increasing the number of MM probes and/or the mis-match positions represented may be used to enhance quantification, accuracy, and confidence.

[00123] As describe above for the PM probes, each candidate MM probe is required to meet the set boundaries of one or more biophysical parameters, such as melting temperature, G+C content, hairpin energy, self-dimers and photolithography synthesis steps. Generally, these parameters are identical or substantially similar to the PM probe biophysical parameters.

[00124] Candidate MM probes that meet the biophysical parameters and optionally, photolithographic parameters above are then screened for the likelihood of cross-hybridization to a target sequence. Usually, a central kmer length is evaluated. For a 25mer candidate MM, a central kmer from the candidate MM, generally a 15mer, 16mer, 17mer, 18mer, or 19mer is compared against the target sequences. A candidate MM probe that contains a central kmer that is identical to a target sequence is eliminated. Next, candidate PM probes for which no suitable candidate MM probes can be identified are also eliminated.

[00125] Each candidate OTU may be evaluated to determine the number of PM probes that are incapable of hybridization to sequences outside the OTU.

[00126] In one embodiment, a pre-partition process is performed. A pre-partition is the largest possible clade (node id) that does not exceed the max partition size. See Figure 6. Typically, useful partition sizes range from about 1,000 to about 8,000 nodes. Any pre-partition that is in a predetermined size range becomes a full-partition. Pre-partitions that are below the minimum partition size are combined into partitions by assembling sister nodes where possible. For example, assume that partitions are allowed to range in size from 1000 to 2000 members. If node A represents 1500 genes and its parent, node B, represents 2500 genes, then node A is considered a pre-partition. If node C is a sibling of node A, and node C represents only 50 genes, then node C is also a pre-partition because moving node C to its parent, node B, would encapsulate more than the maximum partition size of 2000 members.

[00127] To create candidate sequence clusters, transitive sequence clusters are identified using a sliding threshold of two distance matrixes based on either the count of pairwise unique candidate targets or the count of pairwise common candidate targets. Probes prevalent in a large fraction of the sequences in a candidate sequence cluster, e.g., >= 90% of the sequence in the cluster, are identified using the count of sequences containing the PM and the count of sequences with unambiguous data for given PM's locus. For each prevalent probe, a cross-hybridization potential outside the cluster is also tested. All information regarding cluster-PM sets is recorded. Futile clusters are defined as clusters for which only cross- hybridizing probes are identified are removed from the dataset.

[00128] Where necessary, probes that are expected to display some degree of cross-hybridization can be selected. Potentially hybridization-prone probes are constrained to reduce the probability that sequences outside the cluster could hybridize to many of the cluster-specific PM probes. A distribution algorithm can be used to examine a graph of probe-sequence interconnections (edges) and to favor sets of probes that minimize overlapping edges.

[00129] After solutions from all partitions are completed, a global reconciliation of set solutions across partitions is performed. The sequence clusters are locked as OTUs and each cluster's PM probe set is tested for global cross-hybridization against the other remaining PM probe sets. Probes are ranked for utility based on global cross-hybridization patterns.

[00130] The OTUs are assembled and annotated. Typically, each OTU is taxonomically annotated using one term for each rank from domain, kingdom, phylum, sub-phylum, class, sub-class, order, and family. As a result, all the 16S rRNA sequences presented without taxonomic nomenclature and annotated as "environmental samples" or "unclassified" are assigned with taxonomic annotation.

[00131] Each genus-level name recognized by NCBI is read and recorded. For each lineage of taxonomic terms, duplicate adjacent terms are removed; domain-level terms are found by direct pattern match; and phylum-level terms are found as rank immediately subordinate to domain. Order-level terms are found by -ales suffix and family-level terms are found by -eae suffix. If a family level-term is unavailable but a genus is identified (e.g., by match to an accepted list), the genus-level term is used to derive a family level-term. All unrecognized terms found between recognized terms are fit into available ranks (new ranks are not created for extra terms). Empty ranks are filled by deriving root terms from subordinate terms and adding pre-determined suffixes. Finally, the family of an OTU is determined by vote from the family assignment of the sequences. Ties are broken by priority sequences (e.g., sequences derived from genome sequencing projects can be given highest priority). All OTUs within a subfamily are compared by kmer distance among the sequences and OTUs are linked into a subfamily whenever a threshold similarity is observed. Each candidate OTU is evaluated to determine the count of targets which are prevalent across the sequences of the candidate OTU and are not expected to hybridize to sequences outside the OTU.

[00132] Exemplary PM and MM 25mer probes generated using the disclosed algorithms are provided as SEQ ID Nos. 1-50. It should be noted that the above process is applicable to the selection of probes ranging in size from at least 15 nucleotides to at least 200 nucleotides in length and includes probes that are flanked on one or both sides by common or irrelevant sequences, including linking sequences. Furthermore, probes selected by this process can be further processed to yield probes that are smaller than or larger than the original selected probes. For example, probes listed as SEQ ID Nos. 1-50 can be further processed by removing sequences from the 3 'end, 5 'end or both to produce smaller sequences that are identical to at least a portion of the sequence of the 25mers. In other embodiments, larger probes can be generated by incorporating the sequences of probes identified by the disclosed algorithms, i.e., a 25mer probe can be incorporated into a 30mer or larger, 35mer or larger, 40mer or larger, 45mer or larger, 50mer or larger, 55mer or larger, 60mer or larger, 65mer or larger, 70mer or larger, 75mer or larger, 80mer or larger, 85mer or larger or 90mer or larger probe. Additionally, probes listed as SEQ ID Nos. 1- 50 can be shortened on one end and lengthened on the other end to yield probes that range from lOmer to 200mer.

[00133] Probes selected by the above process also include probes that comprise one or more base substitutions, for example uracil in the place of thymine; incorporate one or more base analogs such as nitropyrrole and nitroindole; comprise of one or more sugar substitutions, e.g., ribose in the place of deoxyribose, or any combination thereof. Similarly, probes selected by the process of the invention, may further comprise alternate backbone chemistry, for example, comprising of phosphoramide.

[00134] The size of the collection of putative probes generated by the methodologies of the invention is partially dependent on the length of the particular highly conserved sequence with longer sequences like that of 23 S rRNA gene allowing for a greater number of homologous sequences than a smaller highly conserved sequence such as 16S rRNA gene. In some embodiments, the length of the highly conserved sequence is at least 100 bp, 250 bp, 500 bp, 1,000 bp, 2,000 bp, 4,000 bp, 8,000 bp, 10,000 bp, or 20,000 bp. Additionally, the size of the collection of putative probes generated by the methodologies of the invention is also dependent on the size of the collection of homologous sequences in one or more databases from which sequences are selected for the analysis and generation of probes. Larger collections of homologous sequences, by providing a larger pool of sequences that can be analyzed, allow for the generation of more putative probes. In some embodiments, the starting collection of homologous sequences in one or more databases contains at least 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000 or 10,000,000 sequences. The size of the collection of putative probes is further dependent on the length of the desired probe, because the probe length decreases, as the number of probes that bind to unique sequences increases. Depending on the particular highly conserved sequence, the size of the database and the length of the desired probe, collections of putative probes of at least 100, 1,000, 10,000, 25,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000 or 10,000,000 probes can be generated.

[00135] Detection systems can be constructed from the putative probes generated by the above methods. The detection system can have any number of probes and range from 1 probe to all the probes selected by the methodology. In some embodiments, the detection system comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 36, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 125, 150, 200, 300, 400, 500, 1000, 2,000, 5,000, 10,000, 20,000, 40,000, 50,000, 100,000, 200,000, 500,000, 1,000,000 or 2,000,000 probes. Systems with large number of probes can be used to identify relevant microorganisms in a sample, e.g., an environment or clinical sample, and/or to generate a biosignature. In another embodiment, once relevant microorganisms are known, detection systems with low (e.g., 1-10,000) to medium (e.g., 10,000- 100,000) numbers of probes can be designed for special purpose applications, such as determining one or more specific biosignatures. In some embodiments, knowledge of the identity of relevant microorganisms can be used to select further probes to these microorganisms. If, for instance, five 25mer probes in a first set of probes hybridize to a relevant microorganism, then variants of these five probes can be generated and tested (e.g. in silico) for their binding and biophysical characteristics. Alternately, identification of relevant microorganisms can lead to the generation of new probes that are unlike the probes first used to identify the microorganisms. For example, once novel microorganisms are identified, antibodies can be generated for specific applications.

[00136] To select OTU-specific probes, e.g., oligonucleotide probes specific for organisms that are included within a hierarchical node, additional PM probes can be chosen for each hierarchical node that has more than one child node. To qualify targets for selection to a certain node, a threshold fraction of sequences within a node matching a PM set are enforced. Examples of the threshold fractions included 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%. Coverage of direct sub-nodes (children) is also enforced. For example, each target should be representative of at least 25% of at least one sub- node.

[00137] The specificity of the probes selected by the methods disclosed herein can be validated experimentally in a number of ways. For example, the hybridization signal of a probe in the presence of the target sequence can be measured and compared to the background signal. Target sequences can be derived from one or more pure cultures or from environmental or clinical samples that are known to contain the target sequence. A specific taxa can be identified as present in a sample if a majority (about 70% to about 100%, about 80% to about 100% or about 90% to about 100%) of the probes on the array have a hybridization signal at least about 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, 400 times, 450 times, 500 times, or 1,000 times greater than that of the background. Also, the hybridization signal of the probe can be compared to the hybridization signal of one or more of its mismatch probes. A PM:MM ratio of at least 1.05, 1.10, 1.15, 1.20, 1.25, 1.30, 1.40, 1.45, or 1.50 can indicate that the PM probe can selectively hybridize to its target sequence. An additional way to test the ability of a probe to selectively hybridize to its target is to calculate ^" a pair difference score (d), further explained below. A pair difference score above 1.0 indicates that the probe can selectively hybridize to the target compared to one of its mismatch probes.

[00138] The methods disclosed herein can be used to select and/or utilize organism-specific and/or OTU- specific oligonucleotide probes for biomolecules, such as proteins, DNA, R A, DNA or RNA amplicons, and native rRNA from a target nucleic acid molecule. In some embodiments, probes are designed to be antisense to the native rRNA so that rRNA from samples can be placed on the array to identify actively metabolizing organisms in a sample with no bias from PCR amplification. Actively metabolizing organisms have significantly higher numbers of ribosomes used for the production of proteins, compared to quiescent or dead organisms. Therefore, in some embodiments, the capacity of one or more organisms to make proteins at a particular point in time can be measured. In this way, the array system of the present embodiments can be used to directly identify the metabolizing organisms within diverse communities.

Sample Preparation

[00139] In some embodiments, the sample used can be an ecosystems sample. Ecosystems include microbiomes associated with plants, animals, and humans. Animal and human associated microbiomes include those found in the gastrointestinal tract, respiratory system, nares, urogenital tract, mammary glands, oral cavity, auditory canal, feces, urine, and skin. In some embodiments, the sample can be any kind of clinical or medical sample. For example, samples from blood, urine, feces, nares, the lungs, the gut, other bodily fluids or excretions, materials derived therefrom, or combinations thereof of mammals may be assayed using the array system. Also, the probes selected by the methods disclosed herein and the array system of the present embodiments can be used to identify an infection in the blood of an animal. The probes selected by the methods disclosed herein and the array system of the present embodiments can also be used to assay medical samples that are directly or indirectly exposed to the outside of the body, such as the lungs, ear, nose, throat, the entirety of the digestive system or the skin of an animal. In some embodiments, a sample includes cell culture samples and/or bacterial culture samples. In some embodiments, a sample comprises a pulmonary sample from a subject, including but not limited to sputum, endotracheal aspirate, bronchoalveolar lavage sample, a swab of the endotrachea, materials derived therefrom, or combinations thereof.

[00140] Techniques and systems to obtain genetic sequences from multiple organisms in a sample, such as an ecosystem, medical, or clinical sample, are well known by persons skilled in the art. Many commercially available DNA extraction and purification kits can also be used. Samples, with lower than 2 pg purified DNA may require amplification, which can be performed using conventional techniques known in the art, such as a whole community genome amplification (WCGA) method (Wu et al., Appl. Environ. Microbiol. (2006) 72, 4931-4941). In some embodiments, highly conserved sequences such as those found in the 16S RNA.gene, 23S RNA gene, 5S RNA gene, 5.8S rRNA gene, 12S rR A gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene and nifD gene are amplified. Usually, amplification is performed using PCR, but other types of nucleic acid amplification can be employed. Generally, amplification is performed using a single pair of universal primers specific to a highly conserved sequence. For redundancy or for increased amount of total amplicon concentration, two or more universal probe pairs each specific to a different highly conserved sequence can be used. Representative PCR primers include: bacterial primers 27F and 1492R. In some embodiments, a nucleic acid sample is amplified using a collection of primers each comprising one or more nucleotide positions selected at random from two or more different nucleotides. In some embodiments, primers, nucleotides, or other reagents used in an amplification reaction are labeled to produced labeled amplification products.

[00141] A gel electrophoresis method can also be used to isolate community RNA (McGrath et al., J. Microbiol. Methods (2008) 75: 172-176). Samples with lower than 5 pg purified RNA may require amplification, which can be performed using conventional techniques known in the art, such as a whole community RNA amplification approach (WCRA) (Gao et al., Appl. Environ. Microbiol. (2007) 73:563- 571) to obtain cDNA. In some embodiments, sampling and DNA extraction are conducted as previously described (DeSantis et al., Microbial Ecology, 53(3):371-383, 2007).

[00142] In some embodiments, DNA; total RNA, or a fraction thereof, including rRNA, 16S rRNA, and 23 S rRNA; or combinations thereof are directly labeled and used without any amplification.

Probe Preparation

[00143] Techniques and means for generating oligonucleotide probes to be used on analysis systems, beads or in other systems are well-known by persons skilled in the art. For example, the oligonucleotide probes can be generated by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N- phosphonate or phosphoramidite chemistries (Froehler et al., Nucleic Acid Res. 14:5399-5407 (1986); McBride et al., Tetrahedron Lett. 24:246-248 (1983)). Synthetic sequences are typically between about 10 and about 500 bases in length, more typically between about 15 and about 100 bases, and most preferably between about 20 and about 40 bases in length. In some embodiments, synthetic nucleic acids include non-natural bases, such as, but by no means limited to, inosine. An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., Nature 363:566-568 (1993); U.S. Pat. No. 5,539,083). In some embodiments, at least 10, 25, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 100,000, 200,000, 500,000, 1,000,000 or 2,000,000 probes are included on the array. In further embodiments, each PM probe has one or more corresponding MM probe present on the array. Typically, each PM-MM probe pair is associated with an OTU. In some embodiments, at least 10, 25, 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 100,000, 200,000 or 500,000 probe pairs are placed on the array. Generally, sets of probe pairs have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 probe pairs present.

[00144] In some embodiments, positive control probes that are complementary to particular sequences in the target sequences (e.g., 16S rRNA gene) are used as internal quantification standards (QS) and included in the system. In other embodiments, positive control probes, also known as internal DNA quantification standards (QS) probes are probes that hybridize to spiked-in nucleic acid sequence targets. Usually, the sequences are from metabolic genes. In some embodiments, negative control (NC) probes, e.g., probes that are not complementary or do not appreciably hybridize to sequences in the target sequences (e.g., 16S rRNA gene) are included on the array. Unlike the QS probes, no target material is spiked into the sample mix for the NC probes, prior to sample processing.

Hybridization Platform Fabrication

[00145] In some embodiments, the probes are synthesized separately and then attached to a solid support or surface, which may be made, e.g., from glass, latex, plastic (e.g., polypropylene, nylon, polystyrene), polyacrylamide, nitrocellulose, gel, silicon, or other porous or nonporous material. In some embodiments, the surface is spherical or cylindrical as in the case of microbeads or rods. In other embodiments, the surface is planar, as in an array or microarray. For example, the method described generally by Schena et al, Science 270:467-470 (1995) can be used for attaching the nucleic acids to a surface by printing on glass plates. In other embodiments, typically used for making high-density oligonucleotide arrays, thousands of oligonucleotides complementary to defined sequences are synthesized in situ at defined locations on a surface by photolithographic techniques (see e.g., Fodor et al., 1991, Science 251 :767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91 :5022-5026; Lockhart et al., 1996, Nature Biotechnology 14: 1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270) or other methods for rapid synthesis and deposition of defined oligonucleotides (e.g., Blanchard et al., Biosensors & Bioelectronics 11:687-690). In some of these methods, oligonucleotides (e.g., 25-mers) of known sequence are synthesized directly on a surface such as a derivatized glass slide. Other methods for making analysis systems are also available, e.g., by masking (Maskos and Southern, 1992, Nuc. Acids. Res. 20: 1679-· 1684). Embodiments of the present invention are applicable to any type of array, for example, bead-based arrays, arrays on glass plates or derivatized glass slides as discussed above, and dot blots on nylon hybridization membranes.

[00146] Embodiments of the invention are applicable for use in any analysis system, including but not limited to bead or solution multiplex reaction platforms, or across multiple platforms, for example, Affymetrix GeneChip® Arrays, Illumina BeadChip® Arrays, Luminex xMAP® Technology, Agilent Two-Channel Arrays, MAGIChips (Analysis systems of Gel-immobilized Compounds) or the NanoString nCounter Analysis System. The Affymetrix (Santa Clara, CA, USA) platform DNA arrays can have the oligonucleotide probes (approximately 25mer) synthesized directly on the glass surface by a photolithography method at an approximate density of 10,000 molecules per μπι ² (Chee et al., Science (1996) 274:610-614). Spotted DNA arrays use oligonucleotides that are synthesized individually at a predefined concentration and are applied to a chemically activated glass surface. In general, oligonucleotide lengths can range from a few nucleotides to hundreds of bases in length, but are typically from about lOmer to 50mer, about 15mer to 40mer, or about 20mer to about 30mer in length. Microparticle Systems

[00147] Oligonucleotides produced using techniques known in the art can be built on and/or coupled to microspheres, beads, microbeads, rods, or other microscopic particles for use in arrays, flow cytometry, and other multiplex assay systems. Numerous microparticles are commercially available from about 0.01 to 100 micrometers in diameter. Generally, microparticles from about 0.1-50 μπι, about 1-20 μπι, or about 3-10 μπι are preferred. The size and shapes of microparticles can be uniform or they can vary. In some embodiments, sublots of different sizes, shapes or both are conjugated to probes before combining the sublots to make a final mixed lot of labeled microparticles. The individual sublots can therefore be distinguished and classified based on their size and shape. The size of the microparticles can be measured in practically any flow cytometry apparatus by so-called forward or small-angle scatter light. The shape of the particle can be also discriminated by flow cytometry, e.g., by high-resolution slit-scanning method.

[00148] Microparticles can be made out of any solid or semisolid material including glass, glass composites, metals, ceramics, or polymers. Frequently, the microparticles are polystyrene or latex material, but any type of polymeric material is acceptable including but not limited to brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyacrylamide, polyacrolein, polybutadiene, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, or combinations thereof. Microparticles can be magnetic or non-magnetic and may also have a fluorescent dye, quantum dot, or other indicator material incorporated into the microparticle structure or attached to the surface of the microparticles. Frequently, microparticles may also contain 1 to 30% of a cross-linking agent, such as divinyl benzene, ethylene glycol dimethacrylate, trimethylol propane trimethacrylate, or N,N'methylene-bis-acrylamide or other functionally equivalent agents known in the art.

Target Labeling

[00149] In one embodiment, the nucleic acid targets are labeled so that a laser scanner tuned to a specific wavelength of light can measure the number of fluorescent molecules that hybridized to a specific DNA probe. For arrays, the nucleic acid targets are typically fragmented to between 15 and 100 nucleotides in length and a biotinylated nucleotide is added to the end of the fragment by terminal DNA transferase. At a later stage, the biotinylated fragments that hybridize to the oligonucleotide probes are used as a substrate for the addition of multiple phycoerythrin fluorophores by a sandwich (Streptavidin) method. For some arrays, such as those made by AGILENT or NIMBLEGEN, the purified community DNA can be fluorescently labeled by random priming using the Klenow fragment of DNA polymerase and more than one fluorescent moiety can be used (e.g. controls could be labeled with Cy3, and experimental samples labeled with Cy5 for direct comparison by hybridization to a single analysis system). Some labeling methods incorporate the molecular label into the target during an amplification or enzymatic step to produce multiple labeled copies of the target.

[00150] In some embodiments, the detection system is able to measure the microbial diversity of complex communities without PCR amplification, and consequently, without the inherent biases associated with PCR amplification. Actively metabolizing cells typically contain about 20,000 or more ribosomes for protein assembly compared to quiescent or dead cells that have few. In some embodiments, rRNA can be purified directly from a sample and processed with no amplification step, thereby reducing or avoiding bias caused by preferential amplification of some sequences over others. Thus, in some embodiments, the signal from the analysis system can reflect the true number of rRNA molecules that are present in the samples. This can be expressed as the number of cells multiplied by the number of rRNA copies within each cell. The number of cells in a sample can then be inferred by several different methods, such as, for example, quantitative real-time PCR, or FISH (fluorescence in situ hybridization.). Then the average number of ribosomes within each cell may be calculated.

Hybridization

[00151] Hybridizations can be carried out under conditions well-known by persons skilled in the art. See Rhee et al. (Appl. Environ. Microbiol. (2004) 70:4303-4317) and Wu et al. (Appl. Environ. Microbiol. (2006) 72:4931-4941). The temperature can be varied to reduce or increase stringency and allow the detection of more or less divergent sequences. Robotic hybridization and stringency wash stations can be used to give more consistent results and reduce processing time. In some embodiments, the hybridization and washing process can be accomplished in less than about half an hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours or 24 hours. Generally, hybridization and washing times are reduced for microparticle based detection systems owing to the greater accessibility of the probes to the target molecules. Generally, hybridization times may be reduced for low complexity assays and/or assays for which there is an excess of target analytes.

Signal Quantification

[00152] After hybridization, arrays can be scanned using any suitable scanning device. Non-limiting examples of conventional microarray scanners include GeneChip Scanner 3000 or GeneArray Scanner, (Affymetrix, Santa Clara, CA); and ProScan Array (Perkin Elmer, Boston, MA); and can be equipped with lasers having resolutions of 10 pm or finer. The scanned image displays can be captured as a pixel image, saved, and analyzed by quantifying the pixel density (intensity) of each spot on the array using image quantification software (e.g., GeneChip Analysis system Analysis Suite, version 5.1 Affymetrix, Santa Clara, CA; and ImaGene 6.0, Biodiscovery Inc. Los Angeles, CA, USA). For each probe, an individual signal value can be obtained through imaging parsing and conversion to xy-coordinates. Intensity summaries for each feature can be created and variance estimations among the pixels comprising a feature can be calculated.

[00153] With flow cytometry based detection systems, a representative fraction of microparticles in each sublot of microparticles can be examined. The individual sublots, also known as subsets, can be prepared so that microparticles within a sublot are relatively homogeneous, but differ in at least one distinguishing characteristic from microparticles in any other sublot. Therefore, the sublot to which a microparticle belongs can readily be determined from different sublots using conventional flow cytometry techniques as described in U.S. Patent 6,449,562. Typically, a laser is shined on individual microparticles and at least three known classification parameter values measured: forward light scatter (C which generally correlates with size and refractive index; side light scatter (C ₂ ) which generally correlates with size; and fluorescent emission in at least one wavelength (C ₃ ) which generally results from the presence of fluorochrome incorporated into the labeled target sequence. Because microparticles from different subsets differ in at least one of the above listed classification parameters, and the classification parameters for each subset are known, a microparticle's sublot identity can be verified during flow cytometric analysis of the pool of microparticles in a single assay step and in real-time. For each sublot of microparticles representing a particular probe, the intensity of the hybridization signal can be calculated along with signal variance estimations after performing background subtraction.

Data Processing and Statistical Analysis

[00154] Simultaneous detection of at least 500, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, or more taxa with a high level of confidence can incorporate techniques to de-convolute the signal intensity of numerous probe sets into probability estimates. In some embodiments, the methods, compositions, and systems of the invention enable detection in one assay the presence or absence of a microorganism in a community of microorganisms, such as an environmental or clinical sample when the microorganism comprises less than 0.05% of the total population of microorganisms. In some embodiments, detection includes determining the quantity of the microorganism, e.g., the percentage of the microorganism in the total microorganism population. De-convolution techniques can include the incorporation of NC probe pairs into the analysis system and the use of the data to fit the hybridization signals from the QS probe pairs to the hybridization distribution of the NC probe pairs.

[00155] De-convolution techniques can allow the detection and quantification of nucleic acids in a sample and by inference, the detection and quantification of microorganisms in a sample. In one aspect of the invention, a system is provided for determining the presence or quantity of a microorganism in a sample comprising contacting a sample with a plurality of probes, detecting the hybridization signals of the sample nucleic acids with the probes and de- convoluting the signals to determine the presence, absence and/or quantity of a particular nucleic acid present in a population of nucleic acids where the particular nucleic acid is present at less than 0.01% of the total nucleic acid population. In some embodiments, the particular nucleic acid is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% homologous to other nucleic acids in the population.

[00156] In some embodiments, the data output from an imaged or scanned sample is de-convoluted and analyzed using the following methods. Using an array as an illustrative example, the hybridization signals are converted to xy-coordinates with intensity summaries and variance estimates generated for the pixels using commercial software. The data is outputted using a standard data format like a CEL file (Affymetrix), or a Feature Report file (NimbleGen).

[00157] The hybridization signals undergo background subtraction. Typically, the background intensity is computed independently for each quadrant as the average signal intensity of the least intense 2% of the probes in the quadrant. Other threshold values may also be used, e.g., 0.5%, 1%, 3%, 4%, 5% or 10%. Background intensity is then subtracted from all probes in a quadrant before further computation is performed. This noise removal procedure can be done on a quadrant-by-quadrant basis or across a whole array.

[00158] In some embodiment, array signals are normalized to allow for the comparison of results achieved in different experiments or for the comparison of replicate experiments. Normalization can be achieved by a number of methods. In one embodiment, reproducibility between different probes for the same target are evaluated using a Position Dependent Nearest Neighbor (PDNN) model as described in Zhang L. et al., A model of molecular interactions on short oligonucleotide analysis systems, Nat. Biotechnol. 2003, 21(7):818-821. The PDNN model allows estimation of the sequence specific noise signal and a non-specific background signal, and thus enables estimation of the true intensity for the probes.

[00159] In other embodiments, per-array models of signal and background distributions using responses observed from comparison of the PM and MM probe pairs and the internal DNA quantification standards (QS) probe pairs are created. In one embodiment, the probability that each probe pair is "positive" is determined by calculating a difference score, d, for each probe pair, d may be defined as:

f PM - MM

d = \ -

PM + MM Eqn. 1

wherein:

PM= scaled intensity of the perfect match probe;

MM= scaled intensity of the mismatch probe; and,

d = pair difference score.

The value of d can range from 0 to 2. When PM» MM, the value of d approaches 0; when PM= MM, d = 1 ; and when PM« MM, the value of d approaches 2.

[00160] In some embodiments, the internal DNA quantification standards (QS) and negative control (NC) probe pairs are binned and sorted by attributes of the probes. Examples of the attributes of the probes that can be used in the embodiments of the present invention include, but are not limited to binding energy; base composition, including A+T count, G+C count, and T count; sequence complexity; cross- hybridization binding energy; secondary structure; hair-pin forming potential; melting temperature; and length of the probe. These attributes of the probes may affect hybridization properties of the probes, for example, A+T count may affect hydrogen bonding of the probe, and T count may affect the length and base composition of the fragments produced by the use of DNase. Fragmentation with other enzyme systems may be influenced by the composition of other bases.

[00161] In one embodiment, QS and NC probe pairs are binned and sorted based on the individual probe's A+T count and T count. For each bin (A+T count by T count), the d values from the negative control probes are fit to a normal distribution to derive the scale (mean) and shape (standard deviation). Then, the d values from QS are fit to a gamma distribution to derive scale and shape. For each array, multiple density plots are produced by this process. Two examples of density plots generated from two different probe bins within the same array are shown in Figure 4A-B. The AT count is 14 for the probes represented both figures. The T count is 9 for the probes in Figure 4A, while the T count is 10 for the probes represented in Figure 4B. As these graphs demonstrate, even one extra T, as shown in Figure 4B, can result in appreciable difference in the probe gamma scale parameter. Variations of gamma scale across 79 arrays are shown in Figure 5.

[00162] The parameters derived from gamma and normal distributions are used to derive a pair response score, r, for each probe pair, r is an indicator of the probability that a probe pair is positive, i.e., the probability for a probe pair to be responsive to the target sequence, r may be defined as:

pdf (X = d)

r =

pdf (X = d) + pdf„ _orm (X

Eqn. 2

where:

r = response score to measure the potential that a specific probe pair is binding a target sequence and not a background signal, i.e. the probability of the probe pair being positive for the specific target sequence; pdfy X = d) = probability that d could be drawn from the gamma distribution estimated for the target class ATx Ty;

pdfnorm (X ⁼ d) = probability that d could be drawn from the normal distribution estimated for the target class ATx Ty.

r can range from 0 to 1. r approaches 1 when PM » MM, and r approaches 0 when PM « MM.

[00163] Each set of interrogation probe pairs, e.g., an OTU, can be scored based on pair response scores, cross-hybridization relationships or both. In some embodiments, the system removes data from at least a subset of probe pair sets before making a final call on the presence or quantity of said microorganisms. In one embodiment, the data is removed based on interrogation probe cross hybridization potential. In one embodiment, the scoring of probe pairs is performed by a two-stage process as discussed below. [00164] For example, a two stage analysis can be performed wherein only probe pairs that pass a first stage are analyzed in the next stage. In the first stage, the distribution of r across each set of probe pairs, R, is determined. For each set of probe pairs that is associated with an OTU, the r values of all probe pairs are ranked within the set, and percentage of probe pairs that meet one or more threshold r values are determined. Frequently, three threshold determinations are made at 25% increments across the total range of ranked probe pairs (interquartile Ql, Q2, and Q3); however, any number of threshold determinations or percentage increments can be used. For example, a determination may use one increment at 70% in which probe pairs must pass a threshold value of 80%.

[00165] Typically, to differentiate signal from noise, an OTU is considered to pass Stage 1 if Ql, Q2, and Q3 of the set of probe pairs that is associated with this OTU surpass the threshold of Ql^, Q2 _min , and Q3min, respectively. That is, for an OTU to pass Stage 1 , the r value of 75% of the probe pairs in the set of probe pairs that is associated with that OTU has to be at least Ql _ra m, the r value of 50% of the probe pairs in that set of probe pairs have to be at least 02^, and the r value of 25% of the probe pairs in that set of probe pairs have to be at least Q3 _m j _n . Ql _m i _n is at least about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.82, about 0.84, about 0.86, about 0.88, about 0.90, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, or about 0.99. Q2 _min is at least about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.82, about 0.84, about 0.86, about 0.88, about 0.90, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, or about 0.99. Q3 _min is at least about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.82, about 0.84, about 0.86, about 0.88, about 0.90, about 0.91, about 0.92, about 0.93, about 0.94, about 0.95, about 0.96, about 0.97, about 0.98, about 0.99, about 0.992, about 0.994, about 0.996, about 0.998, or about 0.999. In some embodiments, Ql^, Q2 _mi „, and Q3 _m i„ are determined empirically from spike-in experiments. For example, Ql _m i _n , Q2 _m i„, and Q3 _m i _n are chosen to allow 2 pM amplicon concentration to pass. In one embodiment, Ql _m i„, Q2 _m i _n , and Q3,nin are 0.98, 0.97, and0.82, respectively. These threshold numbers were empirically derived using DNase to fragment the sample sequences. Since DNase has a T- bias, the use of other enzymes may require a shift in the threshold numbers and can be empirically derived.

[00166] In the second stage only the OTUs passing the first are considered as potential sources of cross- hybridization. In some embodiments, for each OTU, only probe-pairs with r > 0.5 (these are the probe pairs considered as to be likely responsive to the target sequence) are further analyzed. In other instances, only probe pairs with r > 0.6, 0.7, 0.8, or 0.9 are considered responsive and are further analyzed. Probe pairs that are unlikely to be responsive (i.e., r < 0.5) are not analyzed further even if their set R, was responsive overall. R ₀ .s represents the subset of probe pairs in which all probe pairs have r > 0.5. Typically, based on the interquartile Ql, Q2 and Q3 values chosen at Stage 1 , most of the probe pairs in the OTUs passing Stage 1 are analyzed. In other embodiments, only the probe-pairs with r > 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, or 0.90 are further analyzed. [00167] For each probe pair in the R ₀ .5 subset, the count of putatively cross-hybridizing OTUs (i.e., the number of OTUs with which the probe pair can cross-hybridize) is determined. In this process, only the OTUs that have passed Stage 1 are considered as potential sources of cross-hybridization. Each probe pair in the R ₀ .5 subset is penalized by dividing its r value by the count of putatively cross-hybridizing OTUs to determine its modified possibility of being positive. The modified possibility of being positive for a probe pair may be represented by a r _x value. r _x may be defined as:

r

r _x =

Eqn. 3

where

Si = Set of OTUs passing Stagel ; and,

S _]x = Set of OTUs passing Stage 1 with cross hybridization potential to the given probe pair

[00168] r _x is proportional to the response of the probe pair and the specificity of the probe pair given the community observed during the first stage. r _x value can range from 0 to 1. For each set of probe pairs associated with an OTU, r _x are calculated for each probe pair and ranked within the set. Interquartile Ql, Q2, Q3 values for the distribution of r _x value in each set of probe pairs are determined. The taxon represented by the OTU is considered to be present if Ql is greater than Q _x Q2 is greater than Q^, or Q3 is greater than Q^. Q _x i is at least about 0.5, at least about 0.55, at least about 0.6, at least about 0.65, at least about 0.7 at least about 0.75, at least at least about 0.8, at least about 0.85, at least about 0.90, at least about 0.95, or at least about 0.97. is at least about 0.5, at least about 0.55, at least about 0.6, at least about 0.65, at least about 0.7 at least about 0.75, at least at least about 0.8, at least about 0.85, at least about 0.90, at least about 0.95, or at least about 0.97. is at least about 0.5, at least about 0.55, at least about 0.6, at least about 0.65, at least about 0.7 at least about 0.75, at least at least about 0.8, at least about 0.85, at least about 0.90, at least about 0.95, or at least about 0.97. In one embodiment, Q _x i is at least 0.66, that is, 75% of the probe pairs in the set of the probe pairs have a r _x value that is at least 0.66.

[00169] A two stage hybridization signal analysis procedure can be performed on hybridization signals from any array or microparticle generated data set, including data generated from the use of any combination of probes selected using the disclosed methodologies. In some embodiments, the second stage of the procedure penalizes probes based on the number of cross-hybridizations, the intensity of the cross-hybridization signals or a combination of the two.

[00170] The method disclosed herein is useful for hierarchical probe set scoring. An OTU may be present at a node at any hierarchical level on a clustering tree. As used herein, an OTU is a group of one or more organisms, such as a domain, a sub-domain, a kingdom, a sub-kingdom, a phylum, a sub-phylum, a class, a sub-class, an order, a sub-order, a family, a subfamily, a genus, a subgenus, a species, or any cluster. In some embodiments, a R0.5 set is collected for each node on the phylogenetic tree and consists of all unique probes from subordinate Resets. For example, for calculating r _x values for probe pairs in a RQ ₅ set for an OTU representing an "order," the count of putatively cross-hybridizing equally-ranked taxa (i.e., "order" node) containing at least one sequence with cross-hybridization potential is used as the denominator in Eqn. 3.

[00171] In some embodiments, the OTUs at the leaf level (e.g., species, sub-genus or genus) are first analyzed. Then each successive level of nodes in the clustering tree is analyzed. In one embodiment, the analysis is performed up to the domain level. In another embodiment, the analysis is performed up to the phylum level. In yet another embodiment, the analysis is performed up to the kingdom level. Penalization for cross-hybridization in Eqn. 3 is only performed for probes on the same taxonomy level. All present taxa are quantified using the mean scaled PM probe intensity after discarding the highest and lowest value of the set R (HybScore). In some embodiments, only taxa present at a first level are analyzed further.

[00172] In some embodiments, a summary abundance score is determined. Corrected abundance scores are created based on G+C content and uracil incorporation. Generally, probes with higher G+C content produce a higher hybridization signal that is typically compensated for correcting the abundance scores.

[00173] The probability of detection for each taxonomic node is determined by summarizing terminal node detection and the breadth of cross-hybridization relationships. Hierarchical probes are scored for evidence of novel organisms based on cluster analysis.

[00174] In some embodiments, the system is capable of analyzing other data in conjunction with that obtained from the analysis of probe hybridization signal strength. In some embodiments, the system can analyze sequencing reaction data including that obtained with high-through put sequencing techniques. In some embodiments, the sequencing data is from same regions of the same highly conserved sequence analyzed by the method disclosed herein using probes.

High Capacity Analysis System Applications

[00175] Numerous subject-derived samples can be assayed to determine the sample's microbiome composition. By having an assay system capable of detecting in a single assay the presence and optionally quantity of at least 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 500,000 or 1,000,000 bacterial or archeal taxa, a complete picture of a microbiotic ecosystem can be achieved quickly and at relatively low cost providing the ability to examine numerous subjects.

[00176] The elucidation of a specific microbiome associated with an ecosystem, animal, human, organ system, condition, and the like allows for the generation of a "signature," "biosignature," or "fingerprint" of the particular environment sampled, terms used interchangeably herein. If the biosignature is from a normal or healthy system or subject, or is from a subject free from a condition under examination, then the associated biosignature can be used as a reference for the comparison of later samples from the same or other subjects to monitor for changes that are associated with an abnormal or unhealthy state or condition. For example, if a later biosignature of a subject shows that the microbiome has shifted away from that associated with a healthy pulmonary status, then preemptive measures could be taken to prevent a continued shift, for example by identifying a disease-related organism or OTU and taking steps to treat it.

[00177] Similarly, a biosignature of an environment can be compared to a biosignature generated from a pool of samples that represent an average or normal biosignature for a population or collection of environments. For example, a sample from an unhealthy individual could be assayed and the microbial biosignature compared to the biosignature seen in a healthy population at large. If one or more microorganisms are detected in the unhealthy individual that are either not seen in the general population or not seen at the same prevalence then therapeutic measures can be taken to selectively eliminate or reduce in number the microorganisms associated with the unhealthy state. For instance, the microflora of the respiratory system can be compared between individuals that suffer from chronic obstructed pulmonary disease (COPD), such as during an exacerbation of the disease, and individuals not suffering from COPD or having COPD that is in remission. If the individuals with exacerbated COPD are shown to have one or more dominant pulmonary microorganisms compared to the other individuals, then an available drug and/or dietary therapy that specifically targets the prevalent, abnormal microorganisms can be administered. Alternatively or additionally, the pulmonary microorganism population in the COPD sufferer can be shifted through the introduction of large numbers of the microorganisms associated with healthy pulmonary status. Once a relationship is known between the prevalence of a particular microorganism or group of microorganisms (e.g. one or more OTUs) and a disease state, then disease progression or treatment response can also be monitored, diagnosed, and/or predicted using the present systems and methods.

[00178] Numerous microbiomes of animals or humans can be analyzed with the present systems and methods including the gut, respiratory system, urogenital tract, mammary glands, skin, oral cavity, auditory canal, and skin. Clinical samples such as blood, sputum, nares, feces, and urine can be used with the method. From the analysis of normal individuals and those suffering from a disease or condition, a large database of fingerprints or biosignatures can be assembled. By comparing the biosignatures between healthy and disease related states, associations can be made as to the influence and importance of individual components of the microbiome.

[00179] Once these associations are made, treatments can be designed and tested to alter the composition of the microbiota seen in the disease state. Additionally, by regularly monitoring the microbial composition of an affected organ system in a diseased individual, disease progress or response to therapy can be observed and if need, additional therapeutic measures taken to alter the microbiome composition to one that is more representative of that seen in a healthy population. [00180] An interesting property of bacteria that has great importance in healthcare, water quality and food safety is quorum sensing. Many bacteria are able to sense the presence of other members of their species or related species and upon reaching a specific density the bacteria start producing various virulence or pathogenicity factors. In other words, the bacteria's gene expression is coordinated as a group. For example, some bacteria produce exopolysaccharides that are known as "slime layers." The secretion of exopolysaccharidse can decrease the ability of white blood cells to phagocytize the microorganisms and make the microorganisms more resistant to therapeutics or cleaning agents. Traditional methodologies require the detection of specific gene expression in order to detect or study quorum sensing and other population induced effects. The present systems and methods can be used to understand the changes that occur in a microbiome that are associated with a given effect such as biofilm formation or toxicity production. One can develop protocols with the present systems and methods to look for and determine conditions that lead to quorum sensing. For example, testing samples at various timepoints and under varying conditions can lead to determining how and when to intervene or reverse population induced expression of virulence or pathogenicity factors.

[00181] In one embodiment, a method is provided to identify a new indicator species for an environmental or health condition with the present systems and methods. The condition can be that of a normal or healthy state. Alternatively, the indicator species can be for an unhealthy or abnormal condition. To indentify a new indicator species, a normal sample is simultaneously assayed to determine the presence or quantity of each OTU associated with all known bacteria, archae, or fungi; this test result is compared to the results achieved in the simultaneous assay of sample from the environment of the condition where the presence or quantity of each OTU associated with all known bacteria, archae, or fungi was determined. Microorganisms that change in abundance at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20- fold, 50-fold or 100-fold, either increasing in abundance or decreasing in abundance represent putative indicator species for a condition.

[00182] In other embodiments, methods are provided for identifying indicators species associated with a disease state, disease progression, treatment regimen, probiotic administration, including progression of disease. In some embodiments the disease is COPD. In some embodiments, the disease relates to a level of COPD activity in a subject, such as a subject having COPD that is not exacerbated (e.g. non- exacerbated COPD), COPD that is exacerbated (e.g. exacerbated COPD), or changing in the level of disease activity (e.g. intermediate COPD exacerbation). Intermediate COPD exacerbation may be indicative of a transition away from an exacerbated state, and may be used as an indication of successful response to therapy. Intermediate COPD exacerbation may be indicative of a transition towards an exacerbated state. Where intermediate COPD exacerbation indicates an onset of COPD exacerbation, intermediate COPD exacerbation can comprise a prediction of the onset of exacerbation of COPD in a subject. A prediction in onset can comprise a prediction in time to onset, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days before an onset of COPD exacerbation; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, or more weeks before an onset of COPD exacerbation. A prediction of onset of COPD exacerbation may be used as a basis for taking medical action, such as therapeutic action, including but not limited to the administration of a therapeutic compound. In other embodiments, methods are provided for monitoring a change in the environment or health status associated with introducing one or more new microorganisms into a community. For example, measures to increase a particular microorganism's percentage of the gut microbiome in an individual, such as feeding a person yogurt or a food supplement containing L. casei, can be monitored using the present methods and systems.

Combined Analysis

[00183] The ability to identify and quantitate the microorganisms in a sample can be combined with a gene expression technology such as a functional gene array to correlate populations with observed gene expression. Similarly, microbiome composition analysis can be correlated with the presence of chemicals, proteins including enzymes, toxins, drugs, antibiotics or other sample constituents. For instance, nucleic acids isolated from a soil sample can be analyzed to elucidate the microbiome composition (e.g. biosignature) and also to identify expressed genes. In the bare, nutrient-poor soils on the Antarctic, this analysis associated chitinase and mannanase expression with Bacteroidetes and CH ₄ - related genes with Alphaproteobacteria. (Yergeau et al., Environmental microarray analyses of Antarctic soil microbial communities. ISME J. 3:340-351, 2009). Significant correlations were also found between taxon abundances and C- and N-cycle gene abundance. From this data, one can predict that certain organisms or groups of organisms are required or account for the majority of an expected or observed enzymatic or degradative process. For example, members of the Bacteroidetes phylum probably degrade the majority of environmental chitin, a major constituent of exoskeletons of insect and arthropods and also of fungi cell walls, at the sample locale.

[00184] This methodology can be used to identify new antibiotic producing organisms, even ones that are unculturable. For instance, soil extracts can be tested for antibiotic activity. If a positive extract is found, a sample of the soil from which a portion was extracted for antibiotic can be analyzed for microbial composition and perhaps gene expression. Major constituents of the microbiome could be correlated with antibiotic activity with the correlation strengthened through gene expression data allowing one to predict that a particular organism or group of organisms is responsible for the observed antibiotic activity.

[00185] In one aspect, the invention provides a method for determining a condition in a sample. In one embodiment, the method comprises a) contacting said sample with a plurality of different probes; b) determining hybridization signal strength for each of said probes, wherein said determination establishes a biosignature for said sample; and, c) comparing the biosignature of said sample to a biosignature for COPD, including COPD exacerbation. In some embodiments, a method is provided for making a prediction about a sample comprising a) determining microorganism population data as the probability of the presence or absence of at least 100 OTUs of microorganisms in said sample; b) determining gene expression data of one or more genes by said microorganisms in said sample and c) using said expression data and population data to make a prediction about said sample. In some embodiments, the prediction entails the identity of a microorganism responsible for a characteristic or condition observed in an environment.

[00186] Other combined analysis methods include the use of a diffusion chamber to retain microorganisms in a sample while one or more constituents or parameters of the sample are changed. For instance, the salinity or pH of the sample can be changed abruptly or gradually over time. Following specific time intervals, the microbiome of the sample in the diffusion chamber can be determined. Microorganisms that cannot tolerate the new environment conditions will die, become reduced in number due to unfavorable conditions or predation, or remain static in their numbers. In contrast, microorganisms that can tolerate the new conditions will at least maintain their number or thrive, perhaps becoming a dominant population. Use of a diffusion chamber coupled with a system capable of detecting the presence or quantity of at least 10,000 OTUs can allow the identification of microorganisms that perish or fail to thrive when placed in a new environment. Such microorganisms are termed "transient", meaning that their percent composition of the microbiome changes quickly. The identification of transient microorganisms can be used to ascertain the time and/or place they were introduced into an environment. Different transient microorganisms can have different half-lives for a particular condition.

[00187] Diffusion chambers can also take the form of a semi-permeable capsule, tube, rod, or sphere or other solid or semi-solid object. A microbiome or a select group of bacteria can be placed inside the capsule, that is then sealed and introduced into an environment for a specified period of time. Upon removal, the capsule is opened and the microbiome or select group of bacteria sampled to ascertain changes in the presence or quantity of the individual constituents. The capsule can be removed once or periodically to sample the microbiome. Alternatively, multiple single use capsules with identical quantities of the microbiome can be used, each one removed and sampled at a different time point. Microbiomes placed in capsules or other semi-permeable containers can be introduced into a living organism, usually through an orifice, to measure changes to the microbiome composition associated with a particular organ or system environment. For example, a semi-permeable capsule or tube containing a microbiome can be introduced into the gastrointestinal system through the mouth or anus. A microbiome from a healthy individual can be introduced in this manner into an unhealthy individual, such as a patient suffering from Crohn's disease or irritable bowel syndrome to ascertain the effect of the unhealthy condition on the normal, healthy individual associated microbiome. In this manner, the efficacy of drug effectiveness and treatment protocols could also be evaluated based on the effects of the gut ecology on a known microbiome. Low Density-Special Purpose Detection Systems

[00188] In some embodiments, probes are selected for constructing special purpose systems including those with arrays or microparticles. Typically, special purpose "low density" systems, are designed for use in a specific environment or for a particular application and usually feature a reduced number of probes, "down-selected" probes, that are specific to organisms that are known or expected to be present in the particular environment, such as associated with a particular biosignature. In some cases the biosignature is fecal contamination. Typically, a low density system comprises no more than 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000 or 10,000 down selected probes or 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500 or 5,000 down selected probes probe pairs (PM and MM probes). In some embodiments, only 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 probes are used per OTU. In further embodiments, only PM probes are used. Generally, these down-selected probes have robust hybridization signals and few or no cross hybridizations. In some embodiments, the collection of down selected probes have a median cross hybridization potential number of less than 20, 15, 10, 8, 7, 6, 5, 4, 3, 2, or 1 per probe. Frequently the down selected probes belong to OTUs that have reduced numbers of probes. In some embodiments, the OTUs of a down select probe collection have a median number of less than 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 probes per OTU. Generally, low density systems feature probes that recognize no more than 10, 25, 50, 100, 250, 500, 1,000, 2,000, or 5,000 taxa. For a set number of probes, a number of design strategies can be employed for low density systems. One approach is to maximize the number of OTUs identified, e.g., use one probe per OTU with no mismatch probes. Another approach is to select probes based on the desired confidence level. Here, multiple probes for each OTU along with corresponding mismatch probes may be required to achieve at least 95% confidence level for the presence and quantity of each OTU. The probes for a particular low density application can be selected by applying a sample from an appropriate environment to a high density analysis system, e.g., a detection system that can in a single assay determine the probability of the presence or quantity of at least 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 250,000, 500,00 or 1,000,000 OTUs of a single domain, such as bacteria, archea, or fungi, or alternatively, for each known OTU of a single domain. Probes associated with prevalent OTUs can be selected for a low density system. Alternately, the OTUs seen jn a sample of interest can be compared with a control sample and shared OTUs subtracted out with the probes associated with the remaining OTUs selected for the low density system. Additionally, probes can be selected based on a change in prevalence of OTUs between the environment of interest and a control environment. For example, OTUs that are at least 2-fold 5-fold, 10- fold, 100-fold or 1,000-fold more abundant in the sample of interest compared to the control sample are included in the down selected probe set. Using this information, a down selected array, bead multiplex system or other low density assay system is designed.

[00189] "Low density" assays systems can be used to identify select microorganisms and determine the percentage composition of various select microorganisms in relation to each other. Low density assay systems can be constructed using probes selected through the disclosed methodologies. These low density systems can identify at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 or more microorganisms. Representative microorganisms to be identified and optionally quantified are listed in Table 7. Additional representative microorganisms to be identified and optionally quantified are listed in Tables 3-5.

Table 7 Representative Microorganisms Recognized by Low Density Assay Systems

[00190] Low density assays systems are useful for numerous environmental and clinical applications. Exemplary applications are listed in Table 7. Medical conditions that can be identified, diagnosed, prognosed, tracked, or treated based on data obtained with a low density system include but are not limited to, cystic fibrosis, chronic obstructive pulmonary disease, Crohn's Disease, irritable bowel syndrome, cancer, rhinitis, stomach ulcers, colitis, atopy, asthma, neonatal necrotizing enterocolitis, obesity, periodontal disease and any disease or disorder caused by, aggravated by or related to the presence, absence or population change of a microorganism. Through the judicious selection of OTUs to be included in a system, the system becomes a diagnostic device capable of diagnosing one or more conditions or diseases with a high level of confidence producing very low rates of false positive or false negative readings.

[00191] In some embodiments, the low density systems also feature confirmatory probes that are specific (complimentary) for genes or sequences expressed in specific organisms. For example, the cqfi virulence gene of Yersinia pestis and the zonula occludens toxin {zof) gene of Vibrio cholerae and also confirmatory probes to Y. pestis or V. cholerae.

Kits

[00192] As used herein a "kit" refers to any delivery system for delivering materials or reagents for carrying out a method of the invention. In the context of assays, such delivery systems include systems that allow for the storage, transport, or delivery of arrays or beads with probes, reaction reagents (e.g., probes, enzymes, etc. in the appropriate containers) and/or supporting materials (e.g., buffers, written instructions for performing the assay etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and/or supporting materials for assays of the invention.

[00193] In one aspect of the invention, kits for analysis of nucleic acid targets are provided. According to one embodiment, a kit includes a plurality of probes capable of determining the presence or quantity over 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 30,000, 40,000 50,000 or 60,000 different OTUs in a single assay. Such probes can be coupled to, for example, an array or plurality of microbeads. In some aspects a kit comprises at least 5, 10, 15, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000 or 2,000,000 interrogation probes selected using the disclosed methodologies and/or for use in the identification and/or comparison of a biosignature of one or more samples.

[00194] The kit can also include reagents for sample processing. In some embodiments, the reagents comprise reagents for the PCR amplification of sample nucleic acids including primers to amplify regions of a highly conserved sequence, such as regions of the 16S rR A gene. In some embodiments, the reagents comprise reagents for the direct labeling of R A, such as rRNA. In further embodiments, the kit includes instructions for using the kit. In other embodiments, the kit includes a password or other permission for the electronic access to a remote data analysis and manipulation software program. Such kits will have a variety of uses, including environmental monitoring, diagnosing disease, monitoring disease progress or response to treatment, and identifying a contamination source and/or the presence, absence, or amount of one or more contaminants.

Computer Implemented Methods

[00195] FIG. 1 illustrates an example of a suitable computing system environment or architecture in which computing subsystems may provide processing functionality to execute software embodiments of the present invention, including probe selection, analysis of samples, and remote networking. The method or system disclosed herein may also operational with numerous other general purpose or special purpose computing system including personal computers, server computers, hand-held or laptop devices, multiprocessor systems, and the like.

[00196] The method or system may be described in the general context of computer-executable instructions, such as program modules, being executed by a computer. The method or system may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network.

[00197] With reference to FIG. 1, an exemplary system for implementing the method or system includes a general purpose computing device in the form of a computer 102.

[00198] Components of computer 102 may include, but are not limited to, a processing unit 104, a system memory 106, and a system bus 108 that couples various system components including the system memory to the processing unit 104. [00199] Computer 102 typically includes a variety of computer readable media. Computer readable media includes both volatile and nonvolatile media, removable and non-removable media and a may comprise computer storage media. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD- ROM, digital versatile disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices.

[00200] The system memory 106 includes computer storage media in the form of volatile and/or nonvolatile memory such as read only memory (ROM) 110 and random access memory (RAM) 112. A basic input/output system 114 (BIOS), containing the basic routines that help to transfer information between elements within computer 102, such as during start-up, is typically stored in ROM 110. RAM 112 typically contains data and/or program modules that are immediately accessible to and/or presently being operated on by processing unit 104. FIG. 1 illustrates operating system 132, application programs 134 such as sequence analysis, probe selection, signal analysis and cross-hybridization analysis programs, other program modules 136, and program data 138.

[00201] The computer 102 may also include other removable/non-removable, volatile/nonvolatile computer storage media. By way of example only, FIG. 1 illustrates a hard disk drive 116 that reads from or writes to non-removable, nonvolatile magnetic media, a magnetic disk drive 118 that reads from or writes to a removable, nonvolatile magnetic disk 120, and an optical disk drive 122 that reads from or writes to a removable, nonvolatile optical disk 124 such as a CD ROM or other optical media. Other removable/non-removable, volatile/nonvolatile computer storage media that can be used in the exemplary operating environment include magnetic tape cassettes, flash memory cards, digital versatile disks, digital video tape, solid state RAM, solid state ROM, and the like. The hard disk drive 116 is typically connected to the system bus 108 through a non-removable memory interface such as interface 126, and magnetic disk drive 118 and optical disk drive 122 are typically connected to the system bus 108 by a removable memory interface, such as interface 128 or 130.

[00202] The drives and their associated computer storage media discussed above and illustrated in FIG. 1, provide storage of computer readable instructions, data structures, program modules and other data for the computer 102. In FIG. 1, for example, hard disk drive 116 is illustrated as storing operating system 132, application programs 134, other program modules 136, and program data 138. A user may enter commands and information into the computer 102 through input devices such as a keyboard 140 and a mouse, trackball or touch pad 142. These and other input devices are often connected to the processing unit 104 through a user input interface 144 that is coupled to the system bus, but may be connected by other interface and bus structures, such as a parallel port or a universal serial bus (USB). A monitor 158 or other type of display device is also connected to the system bus 108 via an interface, such as a video interface or graphics display interface 156. In addition to the monitor 158, computers may also include other peripheral output devices such as speakers (not shown) and printer (not shown), which may be connected through an output peripheral interface (not shown).

[00203] The computer 102 can be integrated into an analysis system, such as a microarray or other probe system described herein. Alternatively, the data generated by an analysis system can be imported into the computer system using various means known in the art.

[00204] The computer 102 may operate in a networked environment using logical connections to one or more remote computers or analysis systems. The remote computer may be a personal computer, a server, a router, a network PC, a peer device or other common network node, and typically includes many or all of the elements described above relative to the computer 102. The logical connections depicted in FIG. 1 include a local area network (LAN) 148 and a wide area network (WAN) 150, but may also include other networks. Such networking environments are commonplace in offices, enterprise-wide computer networks, intranets and the Internet. When used in a LAN networking environment, the computer 102 is connected to the LAN 148 through a network interface or adapter 152. When used in a WAN networking environment, the computer 102 typically includes a modem 154 or other means for establishing communications over the WAN 150, such as the Internet. The modem 154, which may be internal or external, may be connected to the system bus 108 via the user input interface 144, or other appropriate mechanism. In a networked environment, program modules depicted relative to the computer 102, or portions thereof, may be stored in the remote memory storage device.

[00205] In further aspects of the invention, computer-implemented methods are provided for analyzing the presence or quantity of over 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 30,000, 40,000 50,000 or 60,000 different OTUs in a single assay. In one embodiment, computer executable logic is provided for determining the presence or quantity of one or more microorganisms in a sample comprising: logic for analyzing intensities from a set of probes that selectively binds each of at least 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 30,000, 40,000 50,000 or 60,000 unique and highly conserved polynucleotides and determining the presence of at least 97% of all species present in said sample with at least 90%, 95%, 96%, 97%, 98%, 99% or 99.5% confidence level.

[00206] In one embodiment, computer executable logic is provided for determining probability that one or more organisms, from a set of different organisms, are present in a sample. The computer logic comprises processes or instructions for determining the likelihood that individual interrogation probe intensities are accurate based on comparison with intensities of negative control probes and positive control probes; a process or instructions for determining likelihood that an individual OTU is present based on intensities of interrogation probes from OTUs that pass a first quantile threshold; and a process or instructions for penalizing one or more OTUs that have passed the first quantile threshold based on their potential for cross-hybridizing with other probes that have also passed the first quantile threshold.

[00207] In a further embodiment, computer executable logic is provided for determining the presence of one or more microorganisms in a sample. The logic allows for the analysis of a set of at least 1000 different interrogation perfect probes. The logic further provides for the discarding of information from at least 10% of the interrogation perfect match probes in the process of making the determination. In some embodiments, the computer executable logic is stored on computer readable media and represents a computer software product.

[00208] In other embodiments, computer software products are provided wherein computer executable logic embodying aspects of the invention is stored on computer media like hard drives or optical drives. In one embodiment, the computer software products comprise instructions that when executed perform the methods described herein for determining candidate probes.

[00209] In further embodiments, computer systems are provided that can perform the methods of the inventions. In some embodiments, the computer system is integrated into and is part of an analysis system, like a flow cytometer or a microarray imaging device. In other embodiments, the computer system is connected to or ported to an analysis system. In some embodiments, the computer system is connected to an analysis system by a network connection. Figure 2 illustrates one embodiment of a networked system for remote data acquisition or analysis that utilizes a computer system illustrated in Figure 1. In this example, a sample is imaged using a commercially available imaging system and software. The data is outputted using a standard data format like a CEL file (AFFYMETRIX®), or a Feature Report file (NIMBLEGEN®). Then the data is sent to a remote or central location for analysis using a method of the invention. In some embodiments, a standardized analysis is performed providing signal normalization, OTU quantification, and visual analytics. In other embodiments, a customized analysis is performed using a fixed protocol designed for the user's particular needs. In still other embodiments, a user configurable analysis is used, include a protocol that allows for the user to adjust at least one variable before each analysis run.

[00210] After processing, the results are stored in an exchangeable binary format for later use or sharing. Additionally, hybridization scores and OTU probability values may be exported to a tab delimited file or in a format compatible with UniFrac (Lozupone, et al., UniFrac~an online tool for comparing microbial community diversity in a phylogenetic context, BMC Bioinformatics, 7, 371; 2006) for further statistical analysis of the detected sample communities.

[00211] In some embodiments, multiple, interactive views of the data are available, including taxonomic trees, heatmaps, hierarchical clustering, parallel coordinates (time series), bar plots, and multidimensional scaling scatterplots. In some embodiments, the taxonomy tree displays the mean intensities for each detected OTU and displays the leaves of the tree as a heatmap of samples. The tree may be dynamically pruned by filtering OTUs below a certain intensity or probability threshold. Additionally, the tree may be summarized at any level from phylum to subfamily. In other embodiments, the user can hierarchically cluster both OTUs and samples using any of the standard distance and linkage methods from the integrated C Clustering Library (de Hoon, et al., Open source clustering software, Bioinformatics, 20, 1453-1454; 2004), and the resulting dendrograms displayed in a secondary heatmap window. In some embodiments, a third window is provided that displays interactive bar plots of differential OTU intensities to facilitate pairwise comparison of samples. For any two samples, the height of the difference bars displays either the absolute or relative difference in mean intensity between OTUs. The bars may be grouped and sorted along the horizontal axis by any taxonomic rank for easy identification and comparison. Synchronized selection and filtering affords users the unique ability to seamlessly navigate between multiple views of the data. For example, users can select a cluster in the hierarchical clustering window and simultaneously view the selected organisms in the taxonomy tree, immediately revealing both their phylogenetic and environmental relationship. In further embodiments, the data from the analysis system, i.e., analysis system or flow cytometer, can be co-analyzed and displayed with high- throughput sequencing data. In some embodiments, for each organism identified as present in the sample, the user is able to view a list of other environments where the particular organism is found.

[00212] In some embodiments, the screen displays are dynamic and synchronized to allow the selection or filtration of OTUs with changes to any view simultaneously reflected in all other views. Additionally, OTUs confirmed by 16S rR A gene, 18S rR A gene, or 23 S rRNA gene sequencing can be co-displayed in all views.

Business Methods

[00213] In some aspects of the invention, a business method is provided wherein a client images an array or scans a lot of microparticles and sends a file containing the data to a service provider for analysis. The service provider analyzes the data and provides a report to the user in return for financial compensation. In some embodiments, the user has access to the service provider's analysis system and can manipulate and adjust the analysis parameters or the display of the results.

[00214] In another aspect of the invention, a business method is provided wherein a client sends a sample to be processed, imaged or scanned and the data analyzed for the presence or quantity of organisms. The service provider sends a report to the client in return for financial compensation. In some embodiments of the invention, the client has access to a suite of data analysis and display programs for the further analysis and viewing of the data. In further embodiments, the service provider first provides a system or kit to the client. The kit can include a system to assay a majority, or the entirety of the microbiome present or the system can contain "down-selected" probes designed for particular applications. After sample processing and imaging, the client sends the data for analysis by the service provider. In some embodiments of the invention, the client report is electronic. In other embodiments, the client is provided access to a suite of data analysis and display programs for the further viewing, manipulation, comparison and analysis of the data. In some embodiments, the client is provided access to a proprietary database in which to compare results. In other embodiments, the client is provided access to one or more public databases, or a combination of private and public database for the comparison of results. In some embodiments, the proprietary database includes the pooled results (fingerprints, biosignatures) for normal samples or the pooled results from particular abnormal situations such as a disease state. In some embodiments, the biosignatures are continuously and automatically updated upon receipt of a new sample analysis.

[00215] In some embodiments, the database further comprises highly conserved sequence listings. In some embodiments, the database is updated automatically as new sequence information becomes available, for instance, from the National Institutes of Health's Human Microbiome Project. In further embodiments, probe sets are automatically updated based on the new sequence information. Continuous upgrading of the sequence information and refinement of the probe sets allow for increasing accuracy and resolution in determining the composition of microbiomes and the quantity of their individual constituents. In some embodiments, the system compares earlier microbiome biosignatures with later microbiome biosignatures from the same or substantially similar environments and analyzes the changes in probe set composition and hybridization signal analysis parameters for information that is useful in improving or refining the discrimination between related OTUs, identification and quantification of microbiome constituents, or increasing accuracy of the determinations.

[00216] In some embodiments, the database compiles information about specific microbiomes, for example, the microbiota associated with healthy and unhealthy human intestinal microflora including, age, gender and general health status of host, geographical location of host, host's diet (i.e., Western,

Asian or vegetarian), water source, host's occupation or social status, host's housing status.

[00217] In some embodiments, the reference healthy/normal signatures for adults, male and female, and children can be used as benchmarks to identify presymptomatic and symptomatic disease states, response to treatments/therapies, infection, and/or secondary infection associated with disease.

[00218] In some embodiments, the client is provided with a diagnosis or treatment recommendation based on the comparison between the client's sample microbiome and one or more reference microbiome.

EXAMPLES

[00219] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1 : PhyloChip Array Analysis

[00220] Following sample preparation, application, incubation and washing, using standard techniques, PhyloChip G3 arrays were scanned using a GeneArray Scanner from Affymetrix. The scan was captured as a pixel image using standard AFFYMETRIX® software (GCOS vl.6 using parameter: Percentile v6.0) that reduces the data to an individual row in a text-encoded table for each probe. See Table 8.

Table 8 Exemplary Display of Array Data

[INTENSITY]

NumberCells=506944

CellHeader=XY MEAN STDV

NPIXELS

0 0 167.0

47.9 25

1 0 4293.0

1060.2 25

2 0 179.3

43.7 36

3 0 4437.0

681 .5 25

[00221] Each analysis system had approximately 1,016,000 cells, with 1 probe sequence per cell. The analysis system scanner recorded the signal intensity across the array, which ranges from 0 to 65,000 arbitrary units (a.u) in a regular grid with— 30-45 pixels per cell. A 2 pixel margin was used between adjacent cells, leaving approximately 25-40 pixels per probe of usable signal. From these pixels, the AFFYMETRDC® software computed the 75th percentile average pixel intensity (denoted as the "MEAN"), the standard deviation of signal intensity among the about 25-40 pixels (denoted as the "STDV"), and the number of pixels used per cell (denoted as "NPDCELS"). Any cells that had pixels that were three standard deviations apart in signal intensity were classified as outliers.

[00222] The analysis systems were divided into a user-defined number of horizontal and vertical divisions. By default, four horizontal and four vertical divisions were created resulting in 16 regularly spaced sectors for independent background subtraction. The background intensity was computed independently for each quadrant, as the average signal intensity of the least intense 2% (by default) of probes in that quadrant. The background intensity was then subtracted from all probes before further computation.

[00223] The noise value was estimated according to recommendations in the AFFYMETRDC® GeneChip User Guide v3.3. Noise (N) was due to variations in pixel intensity signals observed by the scanner as it read the array surface and was calculated as the standard deviation of the pixel intensities within each of the identified background cells divided by the square root of the number of pixels comprising that cell. The average of the resulting quotients was used for N in the calculations described below:

scalarB

where

B is a background cell

Sj is the standard deviation among the pixels in B

pixi is the count of pixels in B

scalarB is the count of all background cells, cumulative

[00224] The intensities of all probes were then scaled so that the average observed signal intensity of the spiked in probes had a pre-determined signal strength. This was accomplished by finding a scaling factor (Sf) in order to force the mean response of the corresponding PM probes to a target mean using the equation below:

where

e _t = targeted mean intensity (default: 2500)

scalarK _pm = count of probes complementing any spike-in

S _f = scaling factor

[00225] Typically, the pre-determined signal strengths ranged from about 0 to about 65,000. Once the scaling factor was derived, all cell intensities were multiplied by the scaling factor.

[00226] The noise (N) was scaled by the same factor: N _s = N x S _f ; where N _s = scaled noise, N = unsealed noise, and S _f = scaling factor.

[00227] As an alternative or optional step, MM probes with high hybridization signal responses were identified and the robe pair eliminated where: [PM€ 0]v [A4M e O]

where :

PM = scaled intensit of the perfect match probe

MM = scaled intensity of the perfect match probe

srir = reverse standard ratio threshold (default : 1.3)

sdtntr = reverse standard difference threshold multiplier (default : 130)

N* = scaled noise

O = outlier set

The remaining probe pairs were scored by:

where ;

PM = scaled intensity of the perfect match probe

MM = scaled int ensity of the perfect match probe

sri = standard ratio threshold (default : 1 .3 }

sdlm = standard difference threshold multiplier (default : 130)

N = scaled noise

[00228] After classifying an OTU as "present", the present call was propagated upwards through the taxonomic hierarchy by considering any node (subfamily, family, order, etc.) as ^" present' if at least one of its subordinate OTUs was present.

[00229] Hybridization intensity was the measure of OTU abundance and was calculated in arbitrary units for each probe set as the trimmed average (maximum and minimum values removed before averaging) of the PM minus MM intensity differences across the probe pairs in a given probe set.

Example 2: Water Quality Testing— Fecal Contamination Assay

[00230] The dry weather water flow in the lower Mission Creek and Laguna watersheds of Santa Barbara, California, a place associated with elevated fecal indicator bacteria concentrations and human fecal contamination will be sampled with an array of the present invention. The goal is to characterize whole bacterial community composition and biogeographic pattern in an urbanized creek, 2) compare taxa detected by molecular methods to conventional fecal indicator bacteria, and 3) elucidate reliable groups of bacterial taxa to be used in culture-independent community-based fecal contamination monitoring (indicator species for fecal contamination).

[00231] The watersheds flow through an urbanized area of downtown Santa Barbara. Places to be sampled include storm drains, sections of the flowing creek, lagoon (M2, M4) and ocean. Additionally sites include where Old Mission Creek tributary discharges into Mission Creek. The dry creek flow can have many sources including underground springs in the upstream reaches, urban runoff associated with irrigation and washing, groundwater seepage, sump or basement pumps, and potentially illicit sewer connections. Sampling will be done during a period when there will not have been rain for at least 48 hours prior to or during the sampling. Besides the watershed samples, human feces and sewage will be sampled.

Materials and Methods

[00232] Sample description, collection and extraction. Water samples are collected over 3-5 days from a watershed during a period of dry weather. Additionally, fecal samples including human feces sewage inflow are collected. Dissolved oxygen (DO), pH, temperature and salinity are measured along with each sampling. Water samples are filtered in the lab on 0.22 pm filters and extracted for DNA using the UltraClean Water DNA kit (MoBio Laboratories), and archived at -20 °C. Concentrations (by IDEXX) of Total Coliforms, E. coli, and Enterococcus spp., as well as quantitative PCR (qPCR) measurements of Human-specific Bacteroides Marker (HBM) are also performed.

[00233] 16S rRNA gene amplification for analysis system analysis. The 16S rDNA is amplified from the gDNA using non-degenerate Bacterial primers 27F.jgi and 1492R. Polymerase chain reaction (PCR) is carried out using the TaKaRa Ex Taq system (Takara Bio Inc, Japan). The amplification protocol is previously described (Brodie et al., Application of a High Density Oligonucleotide Analysis system Approach to Study Bacterial Population Dynamics during Uranium Reduction and Reoxidation. Applied Environ Microbio. 72:6288-6298, 2006).

[00234] Analysis system processing, and image data analysis. Analysis system analysis is performed using a high-density phylogenetic analysis system (PhyloChip). The protocols are previously reported (Brodie et al., 2006). Briefly, amplicons are concentrated to a volume less than 40μ1 by isopropanol precipitation. The DNA amplicons are fragmented with DNAse, biotin labeled, denatured, and hybridized to the DNA analysis system at 48 °C overnight (> 16 hr). The arrays are subsequently washed and stained. Arrays are scanned using a GeneArray Scanner (Affymetrix, Santa Clara, CA, USA). The CEL files obtained from the Affymetrix software that produces information about the fluorescence intensity of each probe (PM, MM, and control probes) are analyzed using the CELanalysis software designed by Todd DeSantis (LBNL, Berkeley, USA).

[00235] PhyloChip data normalization. All statistical analyses are carried out in R (Team RCD (2008) R: A language and environment for statistical computing)). To correct for variation associated with quantification of amplicon target (quantification variation), and downstream variation associated with target fragmentation, labeling, hybridization, washing, staining and scanning (analysis system technical variation) a two-step normalization procedure is developed: First, for each PhyloChip experiment, a scaling factor best explaining the intensities of the spiked control probes under a multiplicative error model is estimated using a maximum-likelihood procedure. The intensities in each experiment are multiplied with its corresponding optimal scaling factor. In addition, the intensities for each experiment are corrected for the variation in total array intensity by dividing the intensities by its corresponding total array intensity separately for bacteria and archea.

[00236] Statistical Analysis. All statistical analyses were carried out in R. Bray-Curtis distances were calculated using normalized fluorescence intensity with the bcdist function in the ecodist package (Goslee SC & Urban DL (2007) The ecodist package for dissimilarity- based analysis of ecological data. J Stat Softw 22(7): 1-19). Mantel correlation between Bray- Curtis distance matrices of community data, geographical distance and environmental variables are calculated using the mantel function in the vegan package. Pearson's correlation is calculated with 1000 permutations of the Monte Carlo (randomization) test. Non-metric multidimensional scaling (NMDS) is performed using the metaMDS function of the vegan package. A relaxed neighbor-joining tree is generated using Clearcut ( Evans J, Sheneman L, & Foster JA (2006) Relaxed neighbor-joining: a fast distance-based phylogenetic tree. Construction method. J Mol Evol 62:785-792.). Separate clearcut trees are generated for the 'resident' and 'transient' communities for each site. Unweighted UniFrac distances (Lozupone C & Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Applied and Environmental Microbiology 71(12): 8228-8235) are calculated for each of the sites.

PhyloChip derived parameters

[00237] Fecal Taxa. Taxa that are present in all three fecal samples, and in all 27 water samples are tabulated separately. The list of 'Fecal Taxa' is derived by removing those taxa found in all water samples from the taxa that are present in all three fecal samples.

[00238] Transient and resident subpopulations. Taxa that are present in at least one sample from each site across the sampling period are tabulated and variances of the fluorescence intensities for those taxa are generated. The taxa in the top deciles are defined as the 'transient' subpopulation, and taxa in the bottom deciles were defined as the 'resident' subpopuation.

[00239] BBC: A. The number of taxa in the classes of Bacilli, Bacteroidetes, Clostridia, and a - proteobacteria are tallied. The ratio is calculated using the following formula:

_D _ ., . Bac + Bct + CIs

BBC : A =

A

[00240] The count for unique taxa in each of the class is normalized by dividing by the total taxa in each class detected by the analysis system.

[00241] Aligned sequences from published studies are downloaded from Greengenes (DeSantis TZ, et al. (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied and Environmental Microbiology 72(7):5069 ^_, 5072) and re-classified using PhyloChip taxonomy. The counts of unique taxa are tallied for each Bacterial class. BBC:A are calculated using the formula above. If no taxon is detect for a class, the count for the class is set as 0.5.

Resolving community differences among habitats

[00242] Mission Creek samples are delineated into three habitat types: ocean, estuarine lagoon, and fresh water (creeks and storm drain effluent). Bray-Curtis distances of the watershed samples and three fecal samples (two sewage and one human feces) are calculated. Non-metric multidimensional scaling (NMDS) ordination and plotting of the first two axes are used to display the distances between samples. Bacterial communities are clearly separated by habitat types. The drain samples are most similar to the fecal samples. Lagoon samples are most similar to the ocean samples.

[00243] Signature taxa that account for the majority of differences in bacterial communities observed between habitats are identified by comparing the detected taxa at the class level among all habitat types. The number of taxas in each habitat type are divided by the total detected for each sample type to obtain a percent detection. Comparing the fecal samples to samples taken above the urban zone or those from the lagoon or ocean show that there are lower fractions of a-proteobacteria and higher fractions of Bacilli and Clostridia. Moreover, five classes are only detected in the fecal samples: Solibacteres, Unclassified Acidobacteria, Chloroflexi-4, Coprothermobacteria and Fusobacteria. Chloroflexi-3 are only detected in creek samples, and Thermomicrobia, Unclassified Termite group 1, and Unclassified Chloroflexi only in the ocean samples. The top 10 classes with the highest standard deviations across the four habitats are (in descending order): Clostridia, a-^proteobacteria, Bacilli, γ-proteobacteria, β-proteobacteria, Actinobacteria, Flavobacteria, Bacteroidetes, Cyanobacteria, and c-proteobacteria. Of those classes, Clostridia, Bacilli, and Bacteroidetes fractions are higher, but a-proteobacteria fractions were lower. These four taxa can be used as indicators of fecal contamination.

"Transient" and "resident" subpopulations

[00244] Subpopulations of taxa are identified that fluctuate the most between samplings. These are term "transient" populations. Populations that remain stable the sampling period are term "resident" populations. A comparison of taxa found in the "transient" and "resident" subpopulations illustrate differences in community composition from site to site. The six major orders (Enterobacteriales, Lactobacillales, Actinomycetales, Bacteroidales, Clostridiales and Bacillales) of the Fecal Taxa are compared to further dissect the distribution of fecal bacteria over time. The number of transient Enterobacteriales in samples from some sites are extremely high compare to the rest of the sites. While others have high resident subpopulations of Bacillales. Bacteria are identified that are ubiquitous and not affected by changes in the environmental variables measured, as measured by PhyloChip. Bacteria classes that have similar numbers of taxa throughout the watershed and fecal samples included Verrucomicrobiae, Planctomycetacia, α-proteobacteria, Anaerolinaea, Acidobacteria, Sphingobacteria, and Spirochaetes

Bacilli, Bacteroidetes and Clostridia to α-proteobacteria ratio

[00245] Four bacterial classes: Bacilli, Bacteroidetes, Clostridia and a-Proteobacteria are identified as having the highest variance among the habitat types and are further developed as fecal indicators.

[00246] The combined percentage of Bacilli, Bacteroidetes and Clostridia represent about 20-35% of total classes detected in the fecal samples, whereas their percentages at sites with expected cleaner water such as creek, lagoon and ocean are less than 10-15%. At least 45% of the taxa detected in creek water, lagoon and ocean samples are a-Proteobacteria. These microorganisms were classified as Clean Water Taxa as the percentage of Proteobacteria found in fecal samples is significantly lower at about 35-45%. The ratio of Bacilli, Bacteroidetes and Clostridia to a-proteobacteria (BBC:A) for fecal samples is about 3-5-fold higher than the ratios found in other habitat types. The BBC:A ratios are calculated for each site, and exhibit the same pattern as Fecal Taxa counts across all sites with ocean water having the lowest BBC:A of about 0.75-0.90 with samples close to observed sites of fecal contamination at around 1.50 to about 1.90.

[00247] This ratio contains non-coliform associated bacteria, and avoids the potential of false positive fecal detection due to growth of coliforms in the environment. Bacteroidetes and Clostridia are well known fecal-associated anaerobic bacteria. Bacilli are not especially fecal-associated but have been found in aerobic thermophilic swine wastewater bioreactors ( Juteau P, Tremblay D, Villemur R, Bisaillon JG, & Beaudet R (2005) Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste Applied Microbiology and Biotechnology 66:115-122.). Therefore, the presence of Bacilli, Bacteroides and Clostridiales is a good indication of wastewater-, waste treatment-, and human-derived fecal pollution. a-proteobacteria are mostly phototrophic bacteria that are abundant in the environment, and play key roles in global carbon, sulfur and nitrogen cycles. Many a-proteobacteria thrive under low-nutrient conditions, and will be a good proxy for non-fecal bacteria found in non contaminated aquatic environments.

[00248] The results compare well to BBC:A found in other fecal-associated sources that are analyzed by the PhyloChip with mouse cecum, cow colon, sewage contaminated groundwater, human colon, and secondary sewage. These sources have BBC: A of above 1.2. In contrast, anaerobic groundwater has a BBC: A of 0.80-0.99.

[00249] To confirm the value of the BBC:A ratio for detecting fecal contamination, published studies of bacterial communities obtained by sequencing are analyzed. Ratios from mammalian guts, anaerobic digester sludge, ocean, Antarctic lake ice, and drinking water also demonstrate that there are differences between fecal and non-fecal samples. Mammalian gut samples have BBC: A ranging from about 10 to about 260. Anaerobic digester sludge samples have BBC: A of at least 1 to about 10. These results may reflect the highly-selected community in anaerobically-digested waste activated sludge in wastewater treatment. Non- fecal samples have BBC.A from 0 to 0.94. The sequencing results confirm that a BBC:A threshold of 1.0 can be used as a cutoff for identifying fecal pollution in water with values of 1 and above indicating polluted water. This method of calculating a BBC.A value offers numerous advantages including speed, as culturing is not required, greater detection ability as it can detect microorganisms that are currently unculturable and also avoids expense and technical problems associated with PCR cloning and high through-put sequencing.

[00250] The BBC:A ratio can be used to track the source of fecal pollution as the number usually increases in samples obtained from sites closer to a source of fecal pollution.

Example 3: PhyloChip Array

[00251] An array system, "PhyloChip", was fabricated with some of the organism-specific and OTU- specific 16s rRNA probes selected by the methods described herein. The PhyloChip array consisted of 1,016,064 probe features, arranged as a grid of 1,008 rows and columns. Of these features,— 90% were oligonucleotide PM or MM probes with exact or inexact complementarity, respectively, to 16s rRNA genes. Each probe is paired with a mismatch control probe to distinguish target-specific hybridization from background and non-target cross-hybridization. The remaining probes were used for image orientation, normalization controls, or for pathogen-specific signature amplicon detection using additional targeted regions of the chromosome. Each high-density 16s rRNA gene microarray was designed with additional probes that (1) targeted amplicons of prokaryotic metabolic genes spiked into the 16s rRNA gene amplicon mix in defined quantities just prior to fragmentation and (2) were complementary to pre- labelled oligonucleotides added into the hybridization mix. The first control collectively tested the target fragmentation, labeling by biotinylation, array hybridization, and staining scanning efficiency. It also allowed the overall fluorescent intensity to be normalized across all the arrays in an experiment. The second control directly assayed the hybridization, staining and scanning.

[00252] Complementary targets to the probe sequences hybridize to the array and fluorescent signals were captured as pixel images using standard AFFYMETRTX® software (GeneChip Microarray Analysis Suite, version 5.1) that reduced the data to an individual signal value for each probe and was typically exported as a human readable CEL' file. Background probes were identified from the CEL file as those producing intensities in the lowest 2% of all intensities. The average intensity of the background probes was subtracted from the fluorescence intensity of all probes. The noise value (N) was the variation in pixel intensity signals observed by the scanner as it reads the array surface. The standard deviation of the pixel intensities within each of the identified background probe intensities was divided by the square root of the number of pixels comprising that feature. The average of the resulting quotients was used for N in the calculations described below.

[00253] Using previous methods, probe pairs scored as positive are those that meet two criteria: (i) the fluorescence intensity from the perfectly matched probe (PM) was at least 1.3 times greater than the intensity from the mismatched control (MM), and (ii) the difference in intensity, PM minus MM, was at least 130 times greater than the squared noise value (>130 N2). The positive fraction (PosFrac) was calculated for each probe set as the number of positive probe pairs divided by the total number of probe pairs in a probe set. An OTU was considered 'present' when its PosFrac for the corresponding probe set was > 0.92 (based on empirical data from clone library analyses). Replicate arrays cuold be used collectively in determining the presence of each OTU by requiring each to exceed a PosFrac threshold. Present calls were propagated upwards through the taxonomic hierarchy by considering any node (subfamily, family, order, etc.) as 'present' if at least one of its subordinate OTUs was present.

[00254] Hybridization intensity was the measure of OTU abundance and was calculated in arbitrary units for each probe set as the trimmed average (maximum and minimum values removed before averaging) of the PM minus MM intensity differences across the probe pairs in a given probe set. All intensities < 1 were shifted to 1 to avoid errors in subsequent logarithmic transformations. [00255] The analysis methods described in Example 1 can also be applied to a sample that has been applied to the presently described PhyloChip G3 array.

[00256] A Latin Square Validation was carried out on the PhyloChip G3 array. The novel PhyloChip microarray (G3) was manufactured containing multiple probes for each known Bacterial and Archaeal taxon. The array was challenged with triplicate mixtures of 26 organisms combined in known but randomly assigned concentrations spanning over several orders of magnitude using a Latin Square experimental design. Probe-target complexes were quantified by flourescence intensity. To monitor community dynamics within the environment, water samples were taken from the San Francisco Bay (CA) at two time points following a point-source sewage spill. Entire 16S rRNA gene amplicon pools (-100 billion molecules/time point) were evaluated with the array. Three replicates were tested on different days with 78 Latin Square chips and 1 Quantitative Standards only control. The amplicon concentration range was > 4.5 logio- The target concentration was from 0.25 pM to 477.79 pM, increasing 37% per step plus a 0 pM (26 different concentrations). Each chip contained all 26 targets, each with a different concentration 0-66 ng each for 243 ng total spike. The Latin Square matrix is not shown.

[00257] Figure 7 is a chart showing the concentration of 16S amplicon versus PhyloChip response. Concentration is displayed as the log base 2 picomolar concentration within the PhyloChip hybridization chamber. The y-axis is the average of the multiple perfect match probes in the probe set. The vertical error bars denote the standard deviation of 3 replicate trials. The r-squared value over 0.98 indicates that the PhyloChip G3 array is quantitative in its ability to track changes in concentration.

[00258] Figure 8 and 9 shows that model-based detection is an improvement over positive fraction detection of probe sets. Low concentrations (down to 2pM) are differentiated from background in Latin Square.

[00259] Figure 8 is boxplot comparison of the detection algorithm based on pair "response score",r, distribution (novel) versus the positive fraction calculation (previously used with the G2 PhyloChip). In both plots the x-axis is the concentration of the spiked-in 16S amplicon (The arrow begins at 2 picomolar and extends through 500 picomolar). The y-axis ranges between 0 and 1 in both plots. The top plot's y- axis displays the median r score of all the probes within a probe set whereas the bottom plot's y-axis displays the positive fraction from the same data set. At low concentrations, 0.25 pM, both plots show a wide distribution of scores (see long whiskers), at 2pM the top boxplots have short whiskers indicating that multiple measurements using a variety of bacterial and archaeal species all have very similar median r scores. The corresponding concentration on the positive fraction graph has a wide range of positive fraction scores. At nearly all concentrations, the r score outperforms the positive fraction.

[00260] Figure 9 is two graphs that show the comparison of the r score metric versus the pf by receiver operator characteristic (R.O.C) plots. The steeper slope of the top curve compared to the bottom curve demonstrates that the r score metric can differentiate true positives from false positives more efficiently than the pf metric. The grayscale bar indicates the cutoff values (for either r scores or pf) at each point along the curve.

[00261] The validation shows that the novel PhyloChip G3 array is capable of excellent organism detection and quantification in a sample over the prior G2 array.

Example 4: Profiling Bacterial Communities in Patient Samples

[00262] This example describes profiling of airway bacterial communities of a cohort of patients with chronic obstructive pulmonary disease (COPD), using apparatuses and methods of the invention.

Materials and Methods

[00263] Subject selection and sample collection. Potential subjects for this study were screened from a database of airway specimens collected between August 2004 and April 2006 from mechanically ventilated patients admitted to the intensive care units at Moffitt-Long Hospital (University of California, San Francisco), who were enrolled in a study of Pseudomonas aeruginosa in intubated patients (Flanagan et al., 2007, J. Clin Microbiol 45: 1954-1962). Subjects admitted to the ICU with a primary diagnosis of "COPD exacerbation" were identified for inclusion in this study. Available endotracheal aspirates (ETAs) from eight patients were processed for 16S rR A PhyloChip analysis, as described in the herein and also detailed below. To compare results from PhyloChip analysis with conventional clinical cultures, results were obtained for quantitative clinical laboratory bacterial cultures (blood agar, chocolate agar, and EMB media) performed on minibronchoalveolar lavage (m-BAL) airway samples, collected within 1-5 days of the ETA specimen analyzed by PhyloChip, as previously described (Flanagan et al., 2007). In general, m-BALs possess a similar bacterial community composition to that of ETAs obtained concurrently from the same patient. Clinical data (Table 1) were recorded in a secure database, including whether a diagnosis of pneumonia by conventional clinical and radiologic criteria was made during the patient's hospitalization and the time frame between diagnosis and collection of airway samples. The Committee on Human Research at UCSF approved all study protocols, and all patients or their surrogates provided written, informed consent.

Table 1 : Clinical characteristic of subjects and samples

4 78 M 21 piperacillin/tazobactam 31 PA" ^* , SM ^b '

5 86 F 17 levofloxacin 17 PA ^b*

6 85 F 16 doxycycline, 1 PA" ^' moxifloxacin, vancomycin

7 61 M 5 vancomycin, 7 PA" ^* , SA ^b piperacillin tazobactam

8 73 M 3 piperacillin/tazobactam 3 PA", EA ^{b* a} mini-BAL, minibronchoalveolar lavage clinical culture. The most recent, available culture data were obtained from within 1-5 days prior to the endotracheal aspirate sample analyzed by PhyloChip.

"Detected by PhyloChip; ^* >10,000 colony-forming units on quantitative mini-BAL culture. PA,

Pseudomonas aeruginosa; KP, Klebsiella pneumoniae; SA, Staphylococcus aureus; EA, Enterobacter aerogenes; SM, Stenotrophomonas maltophilia; AF, Aspergillus fumigatus.

[00264] DNA extraction, 16S rRNA gene amplification, PhyloChip processing. Total DNA eas extracted from ETAs (200μΙ_.) using a bead-beating step (5.5ms ^'1 for 30 seconds, FastPrep system) (MP Biomedicals, Cleveland, OH) prior to nucleic acid extraction using the Wizard Genomic DNA Purification kit (Promega, Madison, WT). Twelve, 25-cycle PCR reactions, containing 100 ng of DNA, 2.5 mM each of dNTPs, 1.5 μΜ each primer (Bact-27F and Bact-1492R) and 0.02 υ/μί of ExTaq (Takara Bio, Japan), were performed for each sample across a gradient of annealing temperatures (48- 58°C), to maximize the diversity recovered. The resulting PCR products were pooled and gel-purified using the MinElute Gel Extraction kit (Qiagen, Chatsworth, CA). Known concentrations of synthetic 16S rRNA gene fragments and non-16S rRNA gene fragments were spiked into the pooled, purified PCR product, which served as internal standards for normalization. A total of 250ng of purified PCR product per sample was fragmented, biotin-labeled, and hybridized to the microarray as described in Example 2. Washing, staining, and scanning of arrays were conducted according to standard Affymetrix protocols. Background subtraction, noise calculations and scaling were carried out as described in Examples 1 and 2.

[00265] Analysis of PhyloChip data. Detection and quantification criteria for each taxon were applied, as described in Examples 1 and 2. Briefly, probe-pairs consisting of a perfectly matched and mismatched cross hybridization control probe (containing a mismatch at the 13th nucleotide) were scored as positive if they met two criteria: (1) the fluorescence intensity of the perfectly matched probe was >1.3 times greater than that of the mismatched probe, and (2) the difference in intensity in each probe pair was 130 times greater than the squared noise value for that array. The positive fraction (pf ) of probe sets (minimum of 11, median of 24 probe-pairs per taxon) was calculated, and a taxon was considered "present" if the calculated pf was >90%. Statistical analyses were performed in the R environment (www.Rproject.org), using the ecological community analysis package vegan (version 1.16-1). Log-transformed fluorescence intensities were used to calculate Bray-Curtis dissimilarity measures of ecological distance. Nonmetric multidimensional scaling (NMDS), a nonparametric ordination method that maps community relatedness, in this case using the Bray-Curtis distance metric, was used to assess variability in bacterial community structure. The function adonis (Anderson, 2001, Austr. Ecol. 26: 32-46.), which conducts a matrix-based nonparametric analysis of variance, was applied to explore relationships between community composition and clinical variables, including age, gender, number of intubation days, presence of pneumonia, time frame between pneumonia diagnosis and sample collection, antibiotic and corticosteroid treatments, and survival to ICU discharge. Between-group differences in taxon abundance were assessed by two-tailed t- testing with significance adjusted for false discovery using q-values. Taxa exhibiting q values <0.05, a p- value <0.02 and a change of >1,000 fluorescence units (log-fold change in 16S rRNA copy number) were considered statistically and biologically significant. Phylogenetic trees were constructed using representative 16S rRNA sequences from the Greengenes database. A neighbor-joining tree with nearest- neighbor interchange was produced using FastTree (Price et al., 2009, Mol Biol Evol 26: 1641-1650) and uploaded to the Interactive Tree of Life project (itol.embl.de) for annotation.

[00266] Quantitative polymerase chain reaction (Q-PCR). To confirm that changes in array fluorescence intensities were reflective of changes in target organism abundance, triplicate, Q-PCR reactions were performed for selected taxa containing species of interest, using a Stratagene MxP3000 real-time system and the QuantiTect SYBR Green PCR kit (Qiagen). Primers for taxa containing selected species of interest were designed based on PhyloChip probes for the target taxon (Table 2). Reaction conditions included use of 10 ng of DNA extract and 40 cycles of using the annealing temperatures listed in Table 2 for each primer set. Regression analyses of inverse cycle threshold values plotted against PhyloChip fluorescence intensities were determined for each targeted taxon.

Table 2: Primers used for Q-PCR validation of targeted species

Results

[00267] 16S rRNA PhyloChip analysis identified a total of 1,213 bacterial taxa present in airway samples from COPD patients obtained during the course of acute exacerbations (the complete list is provided in Table 3). Despite recent or ongoing exposure to antibiotics across the group, the mean number of taxa detected in each sample was 411 ± 246 taxa (SD). Identified taxa represented a diverse group of species belonging to 38 bacterial phyla and 140 distinct families (Fig. 10A). Bacterial families detected included members of the Pseudomonadaceae, Pasteurellaceae, Helicobacteraceae, Enterobacteriaceae, Comamonadaceae, Burkholderiaceae, and Alteromonadaceae, among many others. In addition, recently described phyla such as the TM7 subgroup of Gram-positive uncultivable bacteria were also detected in the airways of these patients (Table 3).

[00268] Interpersonal variation in bacterial richness (number of taxa detected) was noted across the patient samples (Fig. 10B). Four subjects (patients 1, 4, 5, and 6) exhibited communities with significantly fewer taxa ( p < 0.002) compared with the other four subjects. Patients in which fewer bacterial taxa were detected tended to possess more members of the Pseudomonadaceae. In contrast, members of the Clostridiaceae, Lachnospiraceae, Bacillaceae, and Peptostreptococcaceae were detected more commonly in those patients with richer communities (patients 2, 3, 7). Patient 8 had a large proportion of taxa belonging to the Enterobacteriaceae family, which have been associated with more advanced COPD lung disease. This patient also had radiographic evidence of coexisting bronchiectasis, which was not present in the other patients.

[00269] Given the variation in bacterial richness among samples, which suggested differences in bacterial community composition, NMDS was used to assess variation in bacterial community structure (based on Bray-Curtis dissimilarity measures) across the sample cohort. This revealed two distinct groups of patient samples and confirmed that patient 8 represented a structurally distinct airway community (Fig. 11). Given this separation of subjects based on differing bacterial community structures, the influence of available clinical parameters on community composition was explored. Matrix-based, nonparametric multivariate analysis of variance revealed that across the cohort, the number of elapsed intubation days was significantly associated with bacterial community composition and structure, accounting for the greatest percentage of the observed variability (44%, p < 0.03; Fig. 11). Group 1 patient samples (patients 2, 3, and 7) were characterized by a shorter intubation duration prior to ETA sample collection (<6 days), while those in Group 2 were intubated for significantly longer periods of time ( p < 0.0007; patients 1, 4, 5, and 6; >16 days) and exhibited a significantly less rich community composition compared to that of Group 1 (p < 0.025). Given the community variation between Group 1 and Group 2, differences in the relative abundance of all detected taxa were assessed between the groups, which identified 153 taxa with significantly different relative abundances (Table 4). All of these significant taxa were present in higher abundance in Group 1, the majority of which (77%) belonged to the phylum Firmicutes. These included species such as Lactobacillus kitasatonis, L. perolens, L. sakei, and Bacillus clausii, as well as known pathogenic species such as Streptococcus constellatus, which is a member of the Streptoccocus milleri group (SMG; Table 4). No other clinical variable [including diagnosis of pneumonia (n=6; p < 0.4) or the number of days between pneumonia diagnosis and sample collection (range: 3-52 days; p < 0.6)] demonstrated a significant association with bacterial community composition in this cohort.

[00270] A common core of 75 bacterial taxa representing 27 classified bacterial families was identified in all patients analyzed (Fig. 12). This core group included members of the Pseudomonadaceae, Enterobacteriaceae, Campy lobacteraceae, and Helicobacteraceae, amongst others. In addition, taxa containing species of pathogenic potential, such as Arcobacter cryaerophilus, Brevundimonas diminuta, Leptospira interrogans, as well as P. aeruginosa, were detected in all patients (a complete list of the core taxa is provided in Table 5). The array data for organisms that have previously been associated with COPD airways was also analyzed. Haemophilus influenzae was detected by the array in two subjects (patients 2 and 8), although corresponding m-BAL cultures were negative for this organism. Moraxella catarrhalis was not detected by PhyloChip or culture in any patient sample. However, other phylogenetically related members in the Moraxellaceae family, including Moraxella oblonga, Acinetobacter haemolyticus, and Psychrobacter psychrophilus were identified by the array in 80-100% of subjects (Table 3). Streptococcus pneumoniae was detected in four subjects (patients 2, 3, 7 and 8) despite all m-BALs being culture-negative for this species. Finally, PhyloChip data was also examined for the presence of the atypical bacteria, Mycoplasma pneumoniae and Chlamydophila pneumoniae, which are associated with 3-5% percent of exacerbations. Neither was detected by the PhyloChip, although a related species, Mycoplasma pulmonis, was identified in a single individual (patient 3).

[00271] Quantitative PCR was performed to validate that changes in reported array fluorescence intensities for targeted taxa correlated with changes in target species copy number for a selection of known airway pathogens (P. aeruginosa and Stenotrophomonas maltophilia) and two characteristic gastrointestinal organisms {Campylobacter mucosalis and Helicobacter cetorum). Regression analysis of species abundance determined by Q-PCR and array fluorescence intensity demonstrated strong concordance between the two independent methods for each target organism (Table 9), confirming their presence in these COPD airway samples and the ability of the array to accurately reflect changes in organism relative abundance.

Table 9: Correlation results for species abundance by Q-PCR and 16S rRNA PhyloChip

Example 5 : Airway Microbiota Dynamics during COPD Exacerbations

[00272] This examples describes the characterization of bacterial microbiota of the airway microbiome around the time of acute COPD exacerbations.

[00273] Twenty-five sputum speciments collected over periods before, during, and after acute exacerbations in five patients were analyzed using a PhyloChip microarray, as described herein. Data was analyzed for changes in community diversity, relative abundance of individual OTUs, and association with clinical variables using repeated measures ANOVA, ordination and cluster analysis methods, and Spearman rank correlations, performed using R statistical software.

[00274] Three subjects had exacerbations deemed infectious-related by a clinician and treated with oral steroids plus antibiotics (e.g. azithromycin), while two individuals were treated with oral steroids and decongestants only. Five time points per subject were analyzed, spanning a pre-exacerbation clinically stable period (range: 12 - 126 days before exacerbation onset), at exacerbation before start of new treatments, and post-exacerbation when the subject was clinically stable/improved (range: 25 - 70 days after exacerbation onset). There were significant changes in bacterial diversity over time across all subjects (p = 0.018), which also correlated strongly with clinical symptom scores (Spearman rho=0.5, p < 0.01). Greatest bacterial diversity was observed in samples from at the onset of exacerbation, particularly in subjects with 'infectious-related' events and in whom diversity decreased significantly following antibiotic therapy. Leading up to exacerbation, increased diversity was reflected by all of the following: 1) significant changes in the relative abundance of multiple, existing (i.e. detected in a previous sample from the subject) bacterial OTUs; 2) expansions in existing OTUs or classes of bacteria through the addition of new members; and 3) the appearance of new OTUs not previously detected in the subject. Microarray analysis confirmed culture-based identification and increased abundance of individual species previously implicated in exacerbations. However, members of additional OTUs, including other potentially pathogenic species, demonstrated contemporaneous shifts in relative abundance, including the Enterobacteriaceae family, Actinobacteria and Clostridia classes of bacteria.

[00275] Fig. 20 illustrates a hierarchical cluster analysis of bacterial community composition across samples based on a Bray-Curtis distance metric of dissimilarities in community composition. Samples of subjects 40 and 46 are more closely clustered with themselves. Also illustrated is that samples taken from subject 3 and subject 49 during exacerbation of COPD (3Ex and 49Ex) have different community composition from the pre- and post-exacerbation samples from the respective subjects. In addition, Azithromycin treatment in subject 19 and subject 49 alters overall community composition, which is reflected by less closely-related post-exacerbation samples.

Example 5: Diagnosis of COPD using a Biosignature

[00276] In this example, methods and apparatus of the present invention are used to determine the biosignature and disease state of a subject having an unknown medical condition. A sample can be collected and nucleic acid extracted as described in Example 4. The sample is then tested for the presence, absence, and/or quanitity of OTUs using an array as described in Example 4. The resulting biosignature is then compared to biosignatures for numerous conditions, such as COPD exacerbation, such as a biosignature as determined in Example 4. Based on a comparison of the biosignatures, a clinician makes a diagnosis of healthy, exacerbated COPD, non-exacerbated COPD, or intermediate exacerbated COPD. Based on the diagnosis, and/or on the biosignature, the then prescribes a treatment for the condition, such as a therapeutic compound.

[00277] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

TABLE 3. ALL BACTERIAL TAXA DETECTED BY 16S RRNA PHYLOCHIF IN AIRWAY SAMPLES OF COPD PATIENTS BEING TREATED FOR SEVERE EXACERBATIONS

Phylum Class Order Family S-P Taxon ILP Representative species ⁰

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 508

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf_14 541

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 209

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6425

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6335

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf_6 6345

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6350

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6356

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 6 6359

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 6 6362

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6366

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6368

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6378

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6410

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6412 add mine drainage done TRB82

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf_16 6414 PCE-contaminated site done CLs73

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6421 PCB-polluted soil done WD217

CO Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 6 6423 coal effluent wetland done FW92

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 14 6424 sphagnum peat bog done K-5bl0

Addobacteria Unclassified Unclassified Unclassified sf 1 572 forested wetland done FW144

Addobacteria Addobacteria- Ellin6075/ll-25 Unclassified sf_l 435 anaerobic VC-degrading enrichment done VC47 bacterium

Addobacteria Addobacteria-5 Unclassified Unclassified sf 1 523 soil metagenomic library done 17F9

Addobacteria Addobacteria-4 Ellin6075/ll-25 Unclassified sf 1 790 soil done 11-25

Addobacteria Addobacteria-4 Ifflin6075/ll-25 Unclassified sf_l 87 activated sludge done 2951

Addobacteria Addobacteria-6 Unclassified Unclassified sf 1 350 Mammoth cave done CCM15a

Addobacteria Addobacteria-6 Unclassified Unclassified sf 1 897 Mammoth cave done CCM8b

Addobacteria Addobacteria-6 Unclassified Unclassified sU 1049 soil done CI 12

Addobacteria Solibacteres Unclassified Unclassified sf 1 6426 Great Artesian Basin done Bll

Addobacteria Addobacteria-4 Unclassified Unclassified sf 1 6363 soil done 32-11

Addobacteria Unclassified Unclassified Unclassified sf 1 4222 forested wetland done FW105

Actinobacteria Actinobacteria Addimicrobiales Addimicrobiaceae sf 1 1090

Actirtobacteria Actinobacteria Addimicrobiales Addimicrobiaceae sf 1 1749 forest soil done DUNssu275 (-3A) (OTU#188)

Actinobacteria Actinobacteria Addimicrobiales Addimicrobiaceae sf_l 1856 forested wetland done RCP2-105

Actinobacteria Actinobacteria Addimicrobiales Addimicrobiaceae sf 1 1360 forested wetland done RCP2-103

Actinobacteria Actinobacteria Actinomycetales Addothermaceae sU 1399 uranium mill tailings done Gitt- F-183

Actinobacteria Actinobacteria Actinomycetales Actinomycetaceae sf_l 1684 Varibaculum cambriense str. CCUG 44998

Actinobacteria Actinobacteria Actinomycetales Actinomycetaceae sf_l 1227 Actinomyces naeslundii

Actinobacteria Actinobacteria Actinomycetales Actinomycetaceae sf 1 1672 Actinomyces odontolyticus str. CCUG 28084

Actinobacteria Actinobacteria Actinomycetales Actinosynnemataceae sf 1 1463 Saccharothrix texasensis str. NRRL B-16107T

Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae sf 1 1351 Bifidobacterium psychraerophilum str. T16

Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae sf_l 1967 Bifidobacterium pseudocatenulatum str. JCM1200

Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae sf_l 2040 Bifidobacterium adolescentis str. E- 81074T Actirtobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae sf_l 1109 Bifidobacterium thermacidophilum porcinum subsp. suis str. P3-14 subsp.

Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae sf_l 1987 human subgingival plaque clone CXOIO Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae sf_l 1444 Bifidobacteriaceae genomosp. CI

Actirtobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae sf_l 1835 Bifidobacterium breve str. KB 92

Actinobacteria Actinobacteria Bifidobacteriales Bifidobacteriaceae sf_l 1875

Actinobacteria Actinobacteria Actinomycetales Cellulomonadaceae sf_l 1586 Cellulomonas gelida str. DSM 2011 IT

Actinobacteria Actinobacteria Actinomycetales Cellulomonadaceae sf_l 1748 Beutenbergia cavernosa str. DSM 12333 Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae sf_l 1258 ground water deep-well injection disposal site radioactive wastes Tomsk-7 clone S15A-MN25

Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae sf_l 1800 ground water deep-well injection disposal site radioactive wastes Tomsk-7 clone S15A-

MN100

Actinobacteria Actinobacteria Coriobacteriales Coriobacteriaceae sf_l 1958 Atopobium vaginae VA14183_00

Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae sf_l 1517 Corynebacterium xerosis str. DSM 20743 Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae sf_l 1492 Corynebacterium tuscaniae str. ISS-5309 Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae sf_l 1820 Corynebacterium jeikeium str. ATCC 43734 Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae sf_l 1089 Corynebacterium mucifaciens National

Microbiology Laboratory Special identifier

01-0118

Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae sf_l 1374

Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae sf_l 1428 Corynebacterium simulans National Microbiology

Laboratory Special identifier 00-0186

Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae sf_l 1493 Corynebacterium tuberculostearicum str. CIP102346 Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae sf_l 1803 Cor nebacterium spheniscorum str. CCUG 45512 Actinobacteria Actinobacteria Actinomycetales Derinabacteraceae sf_l 2053 Brackybacterium nesterenkovii str. DSM 9573 Actinobacteria Actinobacteria Actinomycetales Kineosporiaceae sf_l 1598 tichen-dominated Antarctic cryptoendolithic community clone FBP402

Actinobacteria Actinobacteria Actinomycetales Kineosporiaceae sf_l 1961 Kineococcus aurantiacus str. IFO 15268 Actinobacteria Actinobacteria Actinomycetales Microbacteriaceae sf_l 1667 Microbacterium lacticum

Actinobacteria Actinobacteria Actinomycetales Microbacteriaceae sf_l 1197 Arctic sea ice ARK10173

Actinobacteria Actinobacteria Actinomycetales Microbacteriaceae sf_l 1437 freshwater clone SVl-16

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1266 Artkrobacter psychrokctophUus

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1557 Artkrobacter oxydans str. DSM 20119

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf LI 1593 Artkrobacter globiformis

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1610 Artkrobacter sp str. AC-51

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1966 TCE-contaminated site clone ccspost2208 Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1324 glacial ice isolate str. CanDirtyl

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1494 Artkrobacter agilis str. DSM 20550

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1573 Artkrobacter nicotianae str. SB42

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1889 Citricoccus sp. str. 2216.25.22

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 2019 Micrococcus luteus str. HN2-11

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1724 Rotkia mucu ginosa str. DSM

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 2020 Rothia dentocariosa str. ChDC B200

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 2063 Rotkia dentocariosa str. ATCC 17931

Actinobacteria Actinobacteria Actinomycetales Micrococcaceae sf_l 1213 Kocuria roseus

Actinobacteria Actinobacteria Actinomycetales Micromonosporaceae sf_l 1876 Couckioplanes subsp. caeruleus str. IF013939

{continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family s-r Taxon iD ^b Representative species

Actinobacteria Actinobacteria Actinomycetales Mycobacteriaceae sf_l 1175 Mycobacterium cf. xenopi Ήymi_Wue Tb 939/

99' str. HymLWue Tb_939/99

Actinobacteria Actinobacteria Actinomycetales Mycobacteriaceae sf_l 1262 Mycobacterium holsaticum str. 1406

Actinobacteria Actinobacteria Actinomycetales Mycobacteriaceae sf 1 1308 Mycobacterium pyrenworans str. DSM 44605

Actinobacteria Actinobacteria Actinomycetales Mycobacteriaceae sf 1 1365 Mycobacterium chelonae str. CIP 104535T

Actinobacteria Actinobacteria Actinomycetales Mycobacteriaceae sf_l 1650 Mycobacterium tuberculosis str. NCTC 7416

Actinobacteria Actinobacteria Actinomycetales Mycobacteriaceae sf_l 1726 Mycobacterium terrae str. ATCC 15755

Actinobacteria Actinobacteria Actinomycetales Nocardiaceae sU 1834 Nocardia transvalensis str. DSM 43405

Actinobacteria Actinobacteria Actinomycetales Nocardiopsaceae sf 1 1385 Streptomonospora salina str. YIM90002

Actinobacteria Actinobacteria Actinomycetales Promicromonosporaceae sf_l 1671 Cellulosimicrobium cellulans str. NCIMB 11025

Actinobacteria Actinobacteria Actinomycetales Prornkromonosporaceae sf 1 1711 Promicromonospora sukumoe str. DSM 44121

Actinobacteria Actinobacteria Actinomycetales Pseudonocardiaceae sf 1 1863

Actinobacteria Actinobacteria Actinomycetales Pseudonocardiaceae sU 1343 Saccharomonospora azurea str. M.Goodfel

K161 =NA128 (type st

Actinobacteria Actinobacteria Rubrobacterales Rubrobacteraceae sf_l 1551 soil isolate Ellin301

Actinobacteria Actinobacteria Rubrobacterales Rubrobacteraceae sf 1 1739

Actinobacteria Actinobacteria Rubrobacterales Rubrobacteraceae . sf_l 1843 uranium mining waste pile soil sample done

JG30-KF-A23

Actinobacteria Actinobacteria Actinomycetales Sporichthyaceae sf_l 1695 Uchen-dominated Antarctic cryptoendolithic community done FBP417

Actinobacteria Actinobacteria Actinomycetales Streptosporangjaceae sU 1190 Nonomuraea polychroma str. IFO 14345

Actinobacteria Actinobacteria Actinomycetales Thermomonosporaceae sf 1 1741 Actinomadura pelletieri str. IMSNU 22169T

Actinobacteria Actinobacteria Actinomycetales Thermomonosporaceae sf 1 1546 Actinomadura fulvescens str. DSM 43923T

Actinobacteria Actinobacteria Unclassified Unclassified sf_2 1233

Actinobacteria Actinobacteria Unclassified Unclassified sU 1898 termite gut homogenate done Rs-JlO bacterium

Actinobacteria Actinobacteria Unclassified Unclassified sf 1 1367

Actinobacteria Actinobacteria Unclassified Unclassified sf 1 1370 forested wetland done RCPl-37

Actinobacteria Actinobacteria Addimicrobiales Unclassified sf 1 1666

Actinobacteria Actinobacteria Actinomycetales Unclassified sf 4 1337 Sturt arid-zone soil done #0425-2M17

Actinobacteria Actinobacteria Actinomycetales Unclassified sf 3 1486 deep marine sediment done MB-A2-108

Actinobacteria Actinobacteria Unclassified Unclassified sf 1 1676

Actinobacteria Actinobacteria Addimicrobiales Unclassified sf 1 1217 DCP-dechlorinating consortium done SHA-34

Actinobacteria BD2-10 group Unclassified Unclassified sf_2 1652 marine sediment done Bol7

Actinobacteria Actinobacteria Actinomycetales Unclassified sf 3 2045 hypersaline lake done ML602J-44

Actinobacteria Actinobacteria Actinomycetales Unclassified sf_3 1130 Georgenia muralis str. 1A-C

Actinobacteria Actinobacteria Actinomycetales Unclassified sf_3 1687 Jonesia quinghaiensis str. DSM 15701

Actinobacteria Actinobacteria Actinomycetales Unclassified sf 3 1243 termite gut homogenate done Rs-M95 bacterium

Actinobacteria Actinobacteria Actinomycetales Unclassified sf_3 1577 termite gut homogenate done Rs-N91 bacterium

Actinobacteria Actinobacteria Actinomycetales Unclassified sf_3 1405 Arthrobacter ureafaciens str. DSM 20126

AD3 Unclassified Unclassified Unclassified sf_l 2338 uranium mining waste pile soil clone

JG30-KF-C12

Bacteroidetes Bacteroidetes Bacteroidales Bacteroidaceae sf_12 5256 termite gut homogenate done Rs-D38 bacterium

Bacteroidetes Bacteroidetes Bacteroidales Bacteroidaceae sf 12 5320 B cteroides distasonis

Bacteroidetes Bacteroidetes Bacteroidales Bacteroidaceae sf_12 5474 Bacteroides acidofuciens str.A24

Bacteroidetes Bacteroidetes Bacteroidales Bacteroidaceae sf_12 5551 Bacteroides uniformis

Bacteroidetes Bacteroidetes Bacteroidales Bacteroidaceae sf_12 5979 Bacteroides fragilis str. YCH46

Bacteroidetes Flavobacteria Flavobacteriales Blattabacteriaceae sf_l 5828 Blattabacteriutn species

Bacteroidetes Sphingobacteria Sphingobacteriales Crenotrichaceae sf_ll 5334 autotrophic nitrifying biofilm clone NB-11 Bacteroidetes Sphingobacteria Sphingobacteriales Crenotrichaceae sf_ll 5619 anaerobic VC-degrading enrichment clone VCIO bacterium

Bacteroidetes Sphingobacteria Sphingobacteriales Crenotrichaceae sf_ll 5888 penguin droppings sediments clone D9-169 Bacteroidetes Sphingobacteria Sphingobacteriales Crenotrichaceae sf_ll 6123 Flexibacter japonensis str. IFO 16041

Bacteroidetes Sphingobacteria Sphingobacteriales Crenotrichaceae sf_ll 6267 Cilia-respiratory isolate str. 243-54

Bacteroidetes Sphingobacteria Sphingobacteriales Crenotrichaceae sf_ll 6249 Haliscomenobacter hydrossis

Bacteroidetes Sphingobacteria Sphingobacteriales Flamrneovirgaceae sf_5 6084 MicroscUla arenaria str. IFO 15982

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 6079 eynonym: CFB group clone APe4_42 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5367 patient's bronchoalveolar lavage isolate str.

MDA2507 sp.

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5915 groundwater deep-well injection disposal site radioactive wastes Tomsk-7 clone S15A- N27 bacterium

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5997 Flaoobacterium aquatUe

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 6274

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5317 Tenacibaculum maritimum str. IFO 15946 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5991 Tenacibaculum ovoh/ticum str. IAM14318 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 6252 Riftia pachyptila's tube clone R103-B20 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5263 subgingival plaque clone DZ074

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5401 Capnocytophaga gingivalis str. ChDC OS45 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5836 Capnocytophaga granulosa str. LMG 12119; FDC

SD4

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf 1 5423 Aequorivita antarctica str. QSSC9-14

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5942

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5955 Flavobacterium sp. str. V4.MS.29 =MM_-2747 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5971 C tophaga uligtnosa

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5436 Arctic sea ice ARK10004

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5473

Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5267 bacterioplankton clone AEGEAN_179 Bacteroidetes Flavobacteria Flavobacteriales Flavobacteriaceae sf_l 5914 Psychroserpens burtonensis str. S2-64

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 5563 Cytophaga sp. 1-545

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 5542 Cytophaga sp. 1-1787

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 5307 Microscilla sericea str. IFO 16561

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 5357 Flexibacter ruber str. IFO 16677

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 5372

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 5566 Hongiella mannitolworans str. IMSNU 14012

JC2050

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 5667 penguin droppings sediments clone KD6-118 Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 5994 Hymenobacter sp. str. NS/50

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 6124 Flexibacter flexWs subsp. pelliculosus str. IFO

16028 subsp.

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf_19 6297 EBPR sludge lab scale clone HP1A92 Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae sf _20 10311 Cytophaga sp. str. BHI60-57B

Bacteroidetes Bacteroidetes Bacteroidales Porph romonadaceae sf_l 5295 swine intestine done p-987-s962-5

Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae sf_l 5680 termite gut done Rs-106

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family Taxon IE Representative species' ¹

Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae sf_l 5800 Porphyromonas endodontalis str. ATCC 35406

Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae sf_l 5817 termite gut homogenate clone Rs-N56 bacterium

Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae sf_l 5961 cMorobenzene-degrading consortium clone

IA-16

Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae sf_l 5454 Dysgonomonas wimpennyi str. ANFA2

Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae sf_l 5510 sphagnum peat bog clone 26-4b2

Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae sf_l 6012 mouse feces clone Lll-6

Bacteroidetes Bacteroidetes Bacteroidales Porphyromonadaceae sf_l 5460 mouse feces clone F8

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5718 PrevoteUa tantieme str. 29-1

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5437 cow rumen clone BE1

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5916 cow rumen clone BE14

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 6011 rumen clone F24-B03

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 6152 rumen clone RF37

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 6259

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5249 PrevoteUa denticola str. ATCC 35308

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5484 oral periodontitis clone FX046

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5706 oral cavity clone 3.3

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5769 Bacteroidaceae str. A42

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5905 swine intestine clone p-2443-18B5

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5940 PrevoteUa sp. str. E7_34

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 5946 tongue dorsa clone DO027

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 6047 deep marine sediment clone MB-A2-107

Bacteroidetes Bacteroidetes Bacteroidales Prevotellaceae sf_l 6239 tongue dorsa clone DO033

Bacteroidetes Bacteroidetes Bacteroidales Rikenellaceae sf_5 5892 anoxic bulk soil flooded rice microcosm clone

BSV73

Bacteroidetes Sphingobacteria Sphingobacteriales Sphingobacteriaceae sf_l 5513 crevicular epithelial cells done AZ123

Bacteroidetes Sphingobacteria Sphingobacteriales Sphingobacteriaceae sf_l 5913 Sphingobacteriaceae str. Ellinl60

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5573 termite gut homogenate done Rs-D44 bacterium

Bacteroidetes Sphingobacteria Sphingobacteriales Unclassified sf_6 5439 Mono Lake at depth 35 m station 6 July 2000 done ML635J-40 bacterium

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5475 SHA-25 done

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5544 marine? done D3-17

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5783 Mono Lake at depth 35 m station 6 July 2000 done ML635J-15 bacterium

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5874 Paralvmetta palmiformis mucus secretions done

P. palm 53 bacterium

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5890 penguin droppings sediments done KDl-125

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 6046 chlorobenzene-degrading consortium done

ΠΙΒ-1

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5820 cow rumen done BF24

Bacteroidetes Unclassified Unclassified Unclassified sf_l 5745

Bacteroidetes Flavobacteria Flavobacteriales Unclassified sf_3 5248 Delaware River estuary done 1G12

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5355 DCP-dechlorinating consortium done SHA-5

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5481 marine sediment above hydrate ridge done

Hyd89-72 bacterium

Bacteroidetes Unclassified Unclassified Unclassified sf 4 5703

Bacteroidetes Unclassified Unclassified Unclassified sf_4 5785 Mono Lake at depth 35 m station 6 July 2000 clone ML635J-56

Bacteroidetes Unclassified Unclassified Unclassified sf_4 5787 Mono Lake at depth 35 m station 6 July 2000 clone ML635J-1 bacterium

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf_15 5957 Paralvinelk palmiformis mucus secretions clone

P. palm C/20 bacterium

Bacteroidetes Bacteroidetes Bacteroidales Unclassified sf 15 6324 temperate estuarine mud clone KM02

Bacteroidetes Sphingobacteria Sphingobacteriales Unclassified sf_3 6168 Toolik Lake main station at 3 m depth clone

TLMl 1 /TLMdgge04

Bacteroidetes SA1 Unclassified Unclassified sf 1 5951 CFB group clone ML615J-

Bacteroidetes Sphingobacteria Sphingobacteriales Unclassified sf 3 6298 travertine hot spring clone SM1C04

BRC1 Unclassified Unclassified Unclassified sf 2 118 penguin droppings sediments clone KDl-1

BRC1 Unclassified Unclassified Unclassified sf 1 5051 soil clone PBS-HI-24

BRC1 Unclassified Unclassified Unclassified sf 1 5143 soil clone PBS-Π-Ι

Caldithrix Unclassified Caldithrales Caldithraceae sU 91 benzoate-degrading consortium clone BA059

Caldithrix Unclassified Caldithrales Caldithraceae sf 1 2384 saltmarsh clone LCP-89

Chlamydiae Chlamydiae Chlamydiales Chlamydiaceae sf 1 4820 Chlam dophila pneumoniae str. AR39

Chlamydiae Chlamydiae Chlamydiales Farachlamydiaceae sf 1 4964 neutral pH mine biofilm clone 44a-Bl-34

Chlorobi Chlorobia Chlorobiales Chlorobiaceae sf 1 262 ChbroMumferrooxidans DSM 13031 str. ofoX

Chlorobi Chlorobia Chlorobiales Chlorobiaceae sf 1 859 CHorobium phaemribrioides str. 2631

Chlorobi Chlorobia Chlorobiales Chlorobiaceae sf 1 995 Odorobium limicol str. Ml

Chlorobi Unclassified Unclassified Unclassified sf 8 5822 Saltmarsh mud clone K-790

Chlorobi Unclassified Unclassified Unclassified sf 6 5294 Mammoth cave clone CCM9b

Chlorobi Unclassified Unclassified Unclassified sf_9 6146 sludge clone A12b

Chlorobi Unclassified Unclassified Unclassified sf_8 549 benzene-degrading nitrate-reducing consortium clone Cart-N2 bacterium

Chlorobi Unclassified Unclassified Unclassified sf_8 636 benzene-degrading nitrate-reducing consortium clone Cart-N3 bacterium

Chloroflexi Thermomicrobia Unclassified Unclassified sf 1 1041 Antarctic cryptoendolim clone FBP471

Chloroflexi Unclassified Unclassified Unclassified sf_2 818

Chloroflexi Unclassified Unclassified Unclassified sf 5 1051 forest soil clone DUNssu055 (-2B) (OTU#087)

Chloroflexi Anaerolineae Chloroflexi-la Unclassified sf_l 927 Paialvinella palmiformis mucus secretions clone

P. palm C 37 bacterium

Chloroflexi Thermomicrobia Unclassified Unclassified sL2 652 uranium mining waste pile soil sample clone

JG30-KF-CM45

Chloroflexi Anaerolineae Chloroflexi-la Unclassified sf 1 106 DCP-dechlorinating consortium clone SHD-231

Chloroflexi Anaerolineae Unclassified Unclassified sf 9 375 forest soil clone C043

Chloroflexi Anaerolineae Chloroflexi-la Unclassified sf_l 487 thermophilic UASB granular sludge isolate str.

IMO-1 bacterium

Chloroflexi Anaerolineae Unclassified Unclassified sf 9 576 LXT-decWorinating consortium clone SHA-36

Chloroflexi Anaerolineae Chloroflexi-la Unclassified sf 1 583 anaerobic bioreactor clone SH -238

Chloroflexi Anaerolineae Unclassified Unclassified sf_9 72 sediments collected at Charon's Cascade near

Echo River October 2000 clone CCD21

Chloroflexi Anaerolineae Unclassified Unclassified sf_9 727 forest soil clone SQ208

Chloroflexi Unclassified Unclassified Unclassified sf 7 757 LXT-decUorinating consortium clone SHA-8

Chloroflexi Anaerolineae Chloroflexi-la Unclassified sf 1 76 IXT-decMorinating consortium clone SHA-147

Chloroflexi Anaerolineae Chloroflexi-lb Unclassified sf_2 789 travertine hot spring clone SM1D10

Chloroflexi Anaerolineae Unclassified Unclassified sf 9 946 temperate estuarine mud clone KM87

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family Taxon IEr" Representative species'

Chloroflexi Dehalococcoidetes Unclassified Unclassified sf_l 2339 uranium mill tailings soil sample clone

Sh765B-TzT-20 bacterium

Chloroflexi Dehalococcoidetes Unclassified Unclassified sf_l 2367 deep marine sediment clone MB-B2-113

Chloroflexi Dehalococcoidetes Unclassified Unclassified sf_l 2438 deep marine sediment clone MB-A2-110

Chloroflexi Dehalococcoidetes Unclassified Unclassified sf_l 2445 deep marine sediment clone MB-A2-103

Chloroflexi Dehalococcoidetes Unclassified Unclassified sf_l 2485

Chloroflexi Dehalococcoidetes Unclassified Unclassified sf_l 2497 forested wetland clone FW60

Chloroflexi Unclassified Unclassified Unclassified sf_12 2523 sponge clone T IO

Chloroflexi Chloroflexi- Unclassified Unclassified sf_2 2344 forest soil clone C083

Chloroflexi Unclassified Unclassified Unclassified sf_l 2534 forest soil done S085

Coprothermobacteria Unclassified Unclassified Unclassified sf l 751 Coprothermobacter sp. str. Dex80-3

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf 5 4967 Toolik Lake main station at 3 m depth clone

TLM14

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_5 5147 Em iania huxleyi str. Plymouth Marine Laborator

PML 92

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf 5 5112 Cyanidium caldarium str. 14-1-1

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_5 5006

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_ll 5098 Euglena tripteris str. UW OB

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_ll 5123 Lepocinclis fusrformis str. ACOI 1025

~ l Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_5 4966 Adiantum pedatum

<D Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_5 4976 Calypogeia muelleriana

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_13 5000 Mitrastema yamamotoi

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_5 5040 Solatium nigrum

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_5 5182 Epifagus virgmi n— chloroplast

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_5 5183 Pisum sativum— chloroplast

Cyanobacteria Cyanobacteria Chloroplasts Chloroplasts sf_5 5192 Cycas revoluta

Cyanobacteria Unclassified Unclassified Unclassified sf_5 4998

Cyanobacteria Cyanobacteria Thermosynechococcus Unclassified sf_l 5012 Synechococcus sp. str. PCC 6312

Cyanobacteria Unclassified Unclassified Unclassified sf_9 5038 Rumen isolate str. YS2

Cyanobacteria Unclassified Unclassified Unclassified sf_9 5164 termite gut homogenate clone Rs-H34

Cyanobacteria Cyanobacteria Oscillatoriales Unclassified sf_l 5189 Oscillatoria sancta str. PCC 7515

Cyanobacteria Unclassified Unclassified Unclassified sf_5 5015 Chlorogloeopsis fritschii str. PCC 6912

Cyanobacteria Unclassified Unclassified Unclassified sf_5 5030 Hapabsiphon welwitschii

Cyanobacteria Cyanobacteria Nostocales Unclassified sf_l 5057 Nodularia sphaerocarpa str. UTEX B 2093

Cyanobacteria Cyanobacteria Oscillatoriales Unclassified sf_l 5049 Oscillatoria spongeliae str. 520 bg

Cyanobacteria Cyanobacteria Plectonema Unclassified sf_l 5190 Plectonema sp. str. F3

Cyanobacteria Unclassified Unclassified Unclassified sf_8 5206

Demococcus-Thermiis Unclassified Unclassified Unclassified sf_l 178 Thermus sp. str. C4

Demococcus-Triermus Unclassified Unclassified Unclassified sf_l 563 Vulcanithermus mediatlanticus str. TR

Eteinococcus-Thermiis Unclassified Unclassified Unclassified sf _2 637 hypersaline pond clone LA7-B27N

Eteinococcus-Thermus Unclassified Unclassified Unclassified sf_3 920

DSSl Unclassified Unclassified Unclassified sf _2 38 DCP-dechlorinating consortium clone SHA-109

DSS1 Unclassified Unclassified Unclassified sf_l 4405 benzoate-degrading consortium clone BA143

Firmicutes Mollicutes Acholeplasma tales Acholeplasma taceae sf_l 3955 Weeping tea tree witches' broom phytoplasma tree

Firmicutes Mollicutes Acholeplasmatales Acholeplasmataceae sf_l 3961 Clover yellow edge mycoplasma-like organism Firmicutes ollicutes Acholeplasmatales Acholeplasmataceae sf_l 3975 Black raspberry witches' broom phytoplasma str. BRWB witches' broom room

Finnicutes Mollicutes Acholeplasmatales Acholeplasmataceae sU 3976

Firmicutes Mollicutes Acholeplasmatales Acholeplasmataceae sf 1 4044

Firmicutes Mollicutes Acholeplasmatales Acholeplasmataceae sf_l 4045 C inaberry yellows phytoplasma

Firmicutes Mollicutes Acholeplasmatales Acholeplasmataceae sU 4046 Pigeon pea witches' broom mycoplasma-like organism

Firmicutes Bacilli Lactobacillales Aerococcaceae sf 1 3386 feedlot manure clone B87

Firmicutes Bacilli Lactobacillales Aerococcaceae sf 1 3522 Aerococcus viridans

Firmicutes Bacilli Lactobacillales Aerococcaceae sf_l 3631 Abiotrophia defectiva str. GIFU12707

(ATCC49176)

Firmicutes Bacilli Lactobacillales Aerococcaceae sf 1 3870 Abiotrophia para-adiacens str. T T1

Finnicutes Bacilli Lactobacillales Aerococcaceae sf 1 3323 Trichococcus flocculiformis str. DSM 2094 Firmicutes Bacilli Lactobacillales Aerococcaceae sf 1 3326 Nostocoida limicola I str. Ben206

Firmicutes Bacilli Lactobacillales Aerococcaceae sf 1 3504 Marintiactib cMus psychrotolerans str. 021 Firmicutes Bacilli Lactobacillales Aerococcaceae sf 1 3553 Desemzia incerta str. DSM 20581

Firmicutes Bacilli Lactobacillales Aerococcaceae sf 1 3833 Carnobacterium alterfunditum

Firmicutes Bacilli Lactobacillales Aerococcaceae sf 1 3840 Trichococcus pasteurii str. KoTa2

Finnicutes Bacilli Bacillales Alicyclobacillaceae sf 1 3368 geothermal site isolate str. G

Firmicutes Bacilli Bacillales Baciilaceae sf_l 3612 Bacillus schlegelii str. ATCC 43741T

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3419 Bacillus algicola str. MM 3737

Firmicutes Bacilli Bacillales Baciilaceae sf 1 3424 uranium mill tailings clone Gitt- F-76 Firmicutes Bacilli Bacillales Bacillaceae sf 1 3661 Bacillus sp. str. 2216.25.2

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3688 Bacillus sp. str. SAFN-006

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3926 Lake Bogoria isolate 64B4

Firmicutes Bacilli Bacillales Bacillaceae sf 1 234 Bacillus vulcani str. 3S-1

Finnicutes Bacilli Bacillales Bacillaceae sU 283 GeobacUlus thermocatenulatus str. DSM 730 Firmicutes Bacilli Bacillales Bacillaceae sf 1 305 BacUlus thermoleovorans

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3460 GeobacUlus jurassicus str. DS1

Finnicutes Bacilli Bacillales Bacillaceae sf 1 3540 GeobacUlus thermoleovorans str. B23

Firmicutes Bacilli Bacillales Bacillaceae sf_l 3763 GeobacUlus stearothermophUus

Firmicutes Bacilli Bacillales Bacillaceae sf_l 3836 GeobacUlus stearothermophUus str. 46 Finnicutes Bacilli Bacillales Bacillaceae sf 1 385 GeobacUlus stearothermophUus str. ΊΊ0 Firmicutes Bacilli Bacillales Bacillaceae sf 1 462 GeobacUlus thermodenitrificans str. DSM 466 Finnicutes Bacilli Bacillales Bacillaceae sf 1 571 BacUlus caldotenax str. DSM 406

Firmicutes Bacilli Bacillales Bacillaceae sf 1 829 GeobacUlus sp. str. YMTC1049

Firmicutes Bacilli Bacillales Bacillaceae sf_l 3635 BacUlus aeolius str. 4-1

Firmicutes Bacilli Bacillales Bacillaceae sf_l 3827 BacUlus acidogenesis str. 105-2

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3845 hot synthetic compost clone pPD15 Finnicutes Bacilli Bacillales Bacillaceae sf_l 3895 BacUlus sporothermodur ns str. M215 Finnicutes Bacilli Bacillales Bacillaceae sf 1 3283 Bacillus niacini str. EF015566

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3439 BacUlus straits str. 171544

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3589 BacUlus sen galensis str. RS8; OP 106 669 Firmicutes Bacilli Bacillales Bacillaceae sf 1 3650

Firmicutes Bacilli Bacillales Bacillaceae sf 1 1050 BacUlus firmus CV93b

Firmicutes Bacilli Bacillales Bacillaceae sf 1 246 BacUlus sp. 6160m-Cl

Firmicutes Bacilli Bacillales Bacillaceae sU 3550 BacUlus megaterium str. QM B1551

TABLE 3. (CONTINUED)

Phylum Class Order Family S-F* Taxon /D ^b Representative specief

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3345 Bacillus pum us str. S9

Firmicutes Bacilli Bacillales Ba illaceae sf_l 3328 PseudobacUlus cawlinae

Firmicutes Bacilli Bacillales Badllaceae sf 1 3370 Bacillus sp. str. TGS437

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3492 Bacillus subtilis str. IAM 12118T

Firmicutes Bacilli Bacillales Badllaceae sf_l 3579 Bacillus sp. str. TGS750

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3675 Bacillus mojavensis str. M-l

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3706 Bacillus sonorensis str. NRRL B-23155

Firmicutes Bacilli Bacillales Bacillaceae sU 3831 Bacillus licheniformis str. KL-068

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3900 Bacillus licheniformis str. DSM 13

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3909 Bacillus subtilis subsp. Marburg str. 168

Firmicutes Bacilli Bacillales Bacillaceae sf_l 3918 Bacillus subtilis

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3467 Bacillus luciferensis str. LMG 18422

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3489 Bacillus s vestris str. SAFN-010

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3482 garbage compost isolate str. M32

Firmicutes Bacilli Bacillales Bacillaceae sf 1 3383

Firmicutes Bacilli Bacillales Bacillaceae sf_l 3517 Planococcus maritimus str. TF-9

Firmicutes Bacilli Lactobacillales Camobacteriaceae sf 1 3536

Firmicutes Bacilli Lactobacillales Carnobacteriaceae sf 1 3792 Carnobacterium sp. str. D35

Firmicutes Bacilli Bacillales Caryophanaceae sf_l 3285 Caryophanon latum str. DSM 14151

Firmicutes Clostridia Clostridiales Qostridiaceae sf 12 2764

Firmicutes Clostridia Qostridiales Gostridiaceae sf 12 3021 Clostridium caminilhermale str. DVird3

Firmicutes Clostridia Clostridiales Qostridiaceae sf 12 2915 Tepid&acter thalassicus str. SC 562

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 3049 Clostridium paradoxum str. DSM 7308T

Firmicutes Clostridia Clostridiales Qostridiaceae sf 12 3077 Clostridium glycolicum str. DSM 1288

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4156 MCB-contaminated groundwater-treating reactor done RA9C1

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4173 termite gut homogenate done Rs-D81 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4187 Clostridiales oral done P4PB_122 P3

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4278 granular sludge done Rlpl6

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4297

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4300 termite gut done s-060

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4310 termite gut done Rs-056

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4364 oral endodontic infection done MCE3_9

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4406 termite gut homogenate done Rs-J39 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4477 termite gut homogenate done Rs-N85 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4502

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4584 Clostridium papyrosolvens str. DSM 2782

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4614 Clostridium sp. str. JC3

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4622 termite gut clone Rs-L36

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4638

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4554 termite gut done Rs-068

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4180 termite gut homogenate done Rs-M23 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4225 termite gut done Rs-116

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4265 termite gut homogenate done Rs-N70 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf 12 4266 termite gut homogenate done Rs-M86 bacterium

Firmicutes Clostridia Qostridiales Clostridiaceae sf_12 4272 termite gut homogenate clone Rs-M34 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4321 termite gut homogenate clone Rs-C76 bacterium

Firmicutes Clostridia Clostridiales Qostridiaceae sf_12 4357 Lachnospiraceae bacterium 19gly4

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4359 termite gut homogenate clone Rs-C69 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4369 termite gut homogenate clone Rs-N73 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4415 termite gut homogenate clone Rs-K32 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4418 termite gut homogenate clone Rs-H18 bacterium

Firmicutes Clostridia Clostridiales Qostridiaceae sf_12 4475 termite gut homogenate clone Rs-N02 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4507 termite gut homogenate clone Rs-N21 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4524 termite gut clone Rs-093

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4550 swine intestine clone p-320-a3

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4559 cow rumen done BF30

Firmicutes Clostridia Clostridiales Qostridiaceae sf_12 4566 swine intestine done p-2657-65A5

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4582 swine intestine done p-2600-9F5

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4627 termite gut homogenate done Rs-A13 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4306 UASB reactor granular sludge done PD-UASB-4 bacterium

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4607 Clostridium noo i str. NCTC538

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4229

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4551 Clostridium acetobutylicum str. ATCC 824 (T)

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4339 Clostridium chauvoei str. ATCC 10092T

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4598 Clostridium s rdini nse str. DSM 600

Firmicutes Clostridia Qostridiales Qostridiaceae sf_12 4169

Firmicutes Bacilli Lactobacdllales Enterococcaceae sf_l 3261 Enterococcus mundtii str. LMG 10748

Firmicutes Bacilli Lactobacillales Enterococcaceae sf_l 3288 Isolation and identification hyper-ammonia producing swine storage pits manure

Firmicutes Bacilli Lactobacillales Enterococcaceae sf_l 3298 Enterococcus sacch rolyticus str. LMG 11427

Firmicutes Bacilli Lactobacillales Enterococcaceae sf_l 3318 Enterococcus ratti str. ATCC 700914

Firmicutes Bacilli Lactobacillales Enterococcaceae sf 1 3382

Firmicutes Bacilli Lactobacillales Enterococcaceae sf 1 3392 Vagococcus lutrae str. mll34/97/l; CCUG 39187

Firmicutes Bacilli Lactobacillales Enterococcaceae sf_l 3433 Tetragenococcus muriaticus

Firmicutes Bacilli Lactobacillales Enterococcaceae sf_l 3598 Enterococcus solitarius str. DSM 5634

Firmicutes Bacilli Lactobacillales Enterococcaceae sf_l 3680 Melissococcus plutonius str. NCDO 2440

Firmicutes Bacilli Lactobacillales Enterococcaceae sf_l 3713 Enterococcus cecorum str. ATCC43198

Firmicutes Bacilli Lactobacillales Enterococcaceae sf_l 3881 Enterococcus dispar str. LMG 13521

Firmicutes Mollicutes Entomoplasma tales Entomoplasmataceae sf_l 4074 swine intestine done p-2013-s959-5

Firmicutes Mollicutes Anaeroplasma tales Er sipelotrichaceae sf_3 3952 Erysipelothrix rhusiopathiae str. Pecs 56

Firmicutes Mollicutes Anaeroplasma tales Erysipelotrichaceae sf_3 3965 TCE-cxmtaminated site done ccslm238

Firmicutes Mollicutes Anaeroplasma tales Erysipelotrichaceae sf_3 3981 phototrophic sludge done PSB-M-3

Firmicutes Mollicutes Anaeroplasma tales Erysipelotrichaceae sf_3 768

Firmicutes Clostridia Qostridiales Eubacteriaceae sf_l 28 termite gut homogenate done Rs-H81 bacterium

Firmicutes Bacilli Bacillales Halobacillaceae sf_l 3633 Bacillus clausii str. GMBAE 42

Firmicutes Bacilli Bacillales Halobacillaceae sf_l 3344 HalobacUlus yeomjeoni str. MSS-402

Firmicutes Bacilli Bacillales Halobacillaceae sf_l 3488 Hahbacillus salinus str. HSL-3

Firmicutes Bacilli Bacillales Halobacillaceae sf_l 3702 Amphibacillus xylanus str. DSM 6626

Firmicutes Bacilli Bacillales Halobacillaceae sf_l 3756 Salibacillus sp. str. YIM-kknyl6

Firmicutes Bacilli Bacillales Halobacillaceae sf_l 3769 GradHbacillus sp. str. YI -kknyl3

Firmicutes Clostridia Qostridiales Lachnospiraceae sf_5 2698 termite gut homogenate done Rs-B88 bacterium

Firmicutes Clostridia Qostridiales Lachnospiraceae sf_5 2804 Clostridium amygdalinum str. BR-10

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family S-f* Taxon IE Representative species'

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 2961 termite gut homogenate clone Rs-F92 bacterium

Finnicutes Clostridia Clostridiales Lachnospiraceae sf_5 3042 swine intestine clone p-2876-6C5

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 3036 termite gut homogenate clone Rs-F27 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 2668 termite gut homogenate clone Rs-G40 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 3017 termite gut homogenate clone Rs-D48 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 3076 Clostridium nexile

Firmicutes Clostridia Clostridiales Lachnospiraceae sf 5 2825 Butyrivibrio fibrisolvens str. LP1265

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 2834 Butyrivibrio fibrisolvens str. OB156

Finnicutes Clostridia Clostridiales Lachnospiraceae sf_5 2844 Pseudobutyrwibrio ruminis str. pC-XS2

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 3059 Butyrivibrio fibrisolvens str. NCDO 2249

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 2994 tennite gut clone Rs-L15

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 3038 swine intestine clone p-1594-c5

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 3171 Lachnospira pectinoschiza

Finnicutes Clostridia Clostridiales Lachnospiraceae sf_5 2931 tennite gut homogenate clone Rs-G77 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sL5 3060 tennite gut homogenate clone Rs-B14 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 3218 termite gut homogenate clone Rs-N53

Finnicutes Clostridia Clostridiales Lachnospiraceae sL5 2681 tennite gut homogenate clone Rs-K41 bacterium

Finnicutes Clostridia Clostridiales Lachnospiraceae sf_5 4212 tennite gut clone Rs-061

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4273 tennite gut homogenate clone Rs-M14 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sL5 4281 granular sludge clone UASB_brew_B86

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4315 tennite gut homogenate clone Rs-N94 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4331 granular sludge clone UASB_brew_B84

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4335 termite gut homogenate clone Rs-N86 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sf 5 4434 termite gut homogenate done Rs-Kll bacterium

Firmicutes Clostridia Gostridiales Lachnospiraceae sf_5 4510 termite gut homogenate clone Rs-Q53 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4511 ckncm314-B7-17 clone

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4512 granular sludge clone UASB_brew_B25

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4514 termite gut homogenate clone Rs-B34 bacterium

Finnicutes Clostridia Clostridiales Lachnospiraceae sf 5 4533 tennite gut homogenate clone Rs-N06 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4535 ckncm297-Bl-l clone

Firmicutes Clostridia Clostridiales Lachnospiraceae sf 5 4539 termite gut homogenate clone Rs-C61 bacterium

Finnicutes Clostridia Clostridiales Lachnospiraceae sf_5 4540 termite gut homogenate clone Rs-M18 bacterium

Finnicutes Clostridia Clostridiales Lachnospiraceae sf 5 4567 human colonic clone HuCB5

Finnicutes Clostridia Clostridiales Lachnospiraceae sf_5 4571 Faecalibacterium prausnitzii str. ATCC 27766

Firmicutes Clostridia Clostridiales Lachnospiraceae sf 5 4613 rumen clone 3C0d-3

Firmicutes Clostridia Clostridiales Lachnospiraceae sf_5 4623 human colonic clone HuCAl

Firmicutes Clostridia Clostridiales Lachnospiraceae sL5 4525 termite gut homogenate clone Rs-Q18 bacterium

Firmicutes Bacilli LactobariUales Lactobacillaceae sf_l 3330 Lactobacillus kitasatonis str. M9212

Firmicutes Bacilli LactobariUales Lactobacillaceae sf_l 3342 Lactobacillus crispatus str. DSM 20584 T

Firmicutes Bacilli LactobariUales Lactobacillaceae sf_l 3478 Lactobacillus crispatus str. ATCC33197

Finnicutes Bacilli LactobariUales Lactobacillaceae sf_l 3490 Lactobacillus suntoryeus str. LH

Finnicutes Bacilli LactobacUlales Lactobacillaceae sf_l 3618 Lactobacillus jensenii str. KC36b

Firmicutes Bacilli LactobacUlales Lactobacillaceae sf_l 3696 Lactobacillus kalixensis str. Kxl27A2; LMG

22115T; DSM 16043T; CCUG 48459T

Firmicutes Bacilli LactobacUlales Lactobacillaceae sf_l 3395 Lactobacillus reuteri str. DSM 20016 T

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3547 Lactobacillus frumenti str. TMW 1.666

Firrnicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3566 Lactobacillus pontis str. LTH 2587

Firmicutes Bacilli Lactobarillales Lactobacillaceae sLl 3798 Lactobacillus fermentum str. MD-9

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3521 Pediococcus mopinatus str. DSM 20285

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3885 Pediococcus pentosaceus

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3634 Lactobacillus letwazi str. JCL3994

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3767 Lactobacillus euebicus str. CECT 5917T

Firmicutes Bacilli Lactobacillales Lactobacillaceae sLl 3810 Lactobacillus breois

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3829 Lactobacillus paralimentari s str. DSM 13238

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3366 Lactobacillus saerimneri str. GDA154 LMG 22087

DSM 16049 (T); CCUG 48462 (T)

Firmicutes Bacilli Lactobacillales Lactobacillaceae sLl 3418 Lactobacillus subsp. aviarius

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3703 Lactobacillus salwarius str. RA2115

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3914 Lactobacillus c pricasei str. LMK3

Firmicutes Bacilli Lactobacillales Lactobacillaceae sf_l 3821 Lactobacillus casei

Firmicutes Bacilli Lactobacillales Lactobacillaceae sU 3768 Lactobacillus perolens str. L532

Firmicutes Bacilli Lactobacillales Lactobacillaceae sLl 3526 Lactobacillus sakei

Firmicutes Bacilli Lactobacillales Leuconostocaceae sf_l 3497 Weisselh koreensis S-5673

Firmicutes Bacilli Lactobacillales Leuconostocaceae sf_l 3573 Leuconostoc ficulneum str. FS-1

Firmicutes Mollicutes Mycoplasma tales Mycoplasma taceae sf_l 3929 Mycoplasma gypsbengalensis str. Gb-V33

Firmicutes Mollicutes Mycoplasma tales Mycoplasma taceae sf_l 3997 Mycoplasma salivarium str. PG20(T)

Firmicutes Mollicutes Mycoplasmatales Mycoplasma taceae sf_l 4014 Mycoplasma pulmonis str. UAB CI IP

Firmicutes Bacilli Bacillales Paenibacillaceae sf_l 3415 PaenibacUlus nematophUus str. NEMlb

Firmicutes Bacilli Bacillales Paenibacillaceae sU 3630

Firmicutes Bacilli Badllales Paenibacillaceae sU 3595 Pa Aacaius sp. str. MB 2039

Firmicutes Bacilli Bacillales Paenibacillaceae sf_l 3299 Breuibacillus borstelensis str. LMG 15536

Firmicutes Bacilli Bacillales Paenibacillaceae sf_l 3641 Brevibacillus sp. MN 47.2a

Firmicutes Bacilli Bacillales Paenibacillaceae sLl 319 Ammontphilus oxalaticus str. RAOx-FF

Firmicutes Bacilli Bacillales Paenibacillaceae sf_l 625 AmmoniphUus oxalworans str. RAOx-FS

Firmicutes Clostridia Qostridiales Peptococc/ sf_ll 304 Selenomonas rummantium str.JCM6582

Addaminococc

Firmicutes Clostridia Clostridiales Peptococc/ sf_ll 709 Selenomonas ruminantium str.S20

Addaminococc

Firmicutes Clostridia Qostridiales Peptococc/ sf_H 710 Centipeda periodontii str. HB-2

Addaminococc

Firmicutes Clostridia Qostridiales Peptococc/ sLll 131 pig feces done

Addaminococc

Firmicutes Clostridia Qostridiales Peptococc/ sf_ll 181 AUisonelh histaminiformans str. MR2

Addaminococc

Firmicutes Clostridia Qostridiales Peptococc/ sf_ll 59 swine intestine done p-1941-e962-3

Addaminococc

Firmicutes Clostridia Qostridiales Peptococc/ sf_ll 940 VeUloneUa dispar str. DSM 20735

Addaminococc

Firmicutes Clostridia Qostridiales Peptococc/ sUl 1036 Great Artesian Basin done G07

Addaminococc

Firmicutes Clostridia Qostridiales Peptococc/ sf_ll 428 cMorobenzene-degrading consortium done

Addaminococc mA-i

Firmicutes Clostridia Qostridiales Peptococc/ sf_ll 534 chlorobenzene-degrading consortium done

Addaminococc ΠΑ-26

(continued)

TABLB 3. INUED)

Phylum Class Order Family S-F* Taxtm irP Representative species

Firmicutes Clostridia Qostridiales Peptococc/ sf_ll 992 anoxic bulk soil flooded rice microcosm done

Addaminococc BSV43 done

Firmicutes Clostridia Clostridiales Peptococc/ sf_ll 242 Desulfosporosinus arteritis str. DSMZ 7493

Addaminococc

Firmicutes Clostridia Clostridiales Peptococc/ sf_ll 300 berizenenxintaminated groundwater done

Addaminococc ZZ12C8

Firmicutes Clostridia Clostridiales Peptococc/ sf_ll 39 forested wetland done RCP2-71

Addaminococc

Firmicutes Clostridia Clostridiales Peptostreptococcaceae sf_5 2721 termite gut homogenate done Rs-N71 bacterium

Firmicutes Clostridia Clostridiales Peptostreptococcaceae sf_5 2729 DCT-dechlorinating consortium done SHA-58

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf_5 2679 termite gut homogenate done BCf9-13

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf_5 2694 oral periodontitis done FX028

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf 5 2714 termite gut homogenate done Rs-N27 bacterium

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf_5 2913 termite gut homogenate done Rs-N82 bacterium

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf 5 3080 termite gut homogenate done Rs-F43 bacterium

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf_5 3112 Evry municipal wastewater treatment plant done 012C11_B_SDJP15

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf 5 3182 termite gut homogenate done Rs-Q64 bacterium

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf 5 2993 oral done P2PB_46 P3

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf_5 2738 Mogibacterium neglectum str. ATCC 700924

(=P9a-h)

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf 5 2805 oral periodontitis done FX033

Firmicutes Clostridia Clostridiales Peptostreptococcaceae sf_5 2797 Isolation and identification hyper-ammonia producing swine storage pits manure

Firmicutes Clostridia Clostridiales Peptostreptococcaceae sf_5 619 TCE^echlorinating microbial community done

1G

Firmicutes Clostridia Clostridiales Peptostreptococcaceae sf_5 224 Finegoldia magna str. ATCC 29328

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf_5 58 Peptostreptococcus sp. str. E3_32

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf 5 1037 Finegoldia magna

Firmicutes Clostridia Qostridiales Peptostreptococcaceae sf 5 616 PeptoniphUus crimalis str. CCUG 31350

Firmicutes Clostridia Clostridiales Peptostreptococcaceae sf_5 393 Anaerococcus vaginalis str. CCUG 31349

Firmicutes Bacilli Bacillales Sporolactobacillaceae sf 1 3365 Bacillus sp. done ML615J-19

Firmicutes Bacilli Bacillales Sporolactobadllaosae sf 1 3747 Bacillus sp. str. C-59-2

Firmicutes Bacilli BacUlales Staphylococcaceae sU 3258 Staphylococcus auricukris str. MAFF911484

ATCC33753T

Firmicutes Bacilli Bacillales Staphylococcaceae sf 1 3284

Firmicutes Bacilli Bacillales Staphylococcaceae sf 1 3545

Firmicutes Bacilli Bacillales Staphylococcaceae sf 1 3569 Staphylococcus saprophyticus

Firmicutes Bacilli Bacillales Staphylococcaceae sf_l 3585

Firmicutes Bacilli Bacillales Staphylococcaceae sf_l 3592 Staphylococcus caprae str. DSM 20608

Firmicutes Bacilli Bacillales Staphylococcaceae sf_l 3605

Firmicutes Bacilli Bacillales Staphylococcaceae sf 1 3628 Staphylococcus haemolyticus str. CCM2737

Firmicutes Bacilli Bacillales Staphylococcaceae sf_l 3638 Staphylococcus sp str. AG-30

Firmicutes Bacilli Bacillales Staphylococcaceae sf 1 3654 Staphylococcus pettenkqjeri str. B3117

Firmicutes Bacilli Bacillales Staphylococcaceae sf_l 3684 Staphylococcus sciuri

Firmicutes Bacilli Bacillales Staphylococcaceae sf_l 3794

Firmicutes Bacilli Badllales Staphylococcaceae sf_l 3822 Staphylococcus succmus str. SB72

Firmicutes Bacilli Bacillales Staphylococcaceae sf_l 3494 Micrococcus luteus B-P 26

Firmicutes Bacilli Badllales Staphylococcaceae sf_l 3865 Macrococcus lamae str. CCM 4815

Firmicutes Bacilli Bacillales Staphylococcaceae sf_l 3432 deep-sea sediment isolate str. P_wp0225

Firmicutes Bacilli LactobariUales Strep tococcaceae sf_l 3722 Lactococcus Π1403 subsp. lactis str. IL1403

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3869 Streptococcus equi subsp. zooepidemicus str.

Tokyol291 subsp.

Firmicutes Bacilli Lactobadllales Streptococcaceae sf_l 3699 Streptococcus agakctiae str. 2603V/R

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3907 aortic heart valve patient with endocarditis done v6

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3250 Streptococcus boots str. B315

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3253 derived cheese sample done 32CR

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3313 Streptococcus salivarius str. ATCC 7073

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3397 Streptococcus macedonicus str. ACA-DC 206

LAB617

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3422 Streptococcus thermopMus str. DSM 20617

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3543

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3588 Streptococcus dovmei str. ATCC 33748

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3906 Streptococcus bovis str VTCC 43143

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3499 Streptococcus constellatus str. ATCC27823

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3446 Streptococcus bovis str. HJ50

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3560 Streptococcus gallinaceus str. CCUG 42692

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3753 Streptococcus suis str. 8074

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3251 Streptococcus cristatus str. ATCC 51100

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3287 tongue dorsum scrapings done FP015

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3290 Streptococcus mitis str. Sm91

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3629 Streptococcus mutans str. UA96

Firmicutes Bacilli Lactobacillales Streptococcaceae sf_l 3685 Streptococcus gordonii str. ATCC 10558

Firmicutes Clostridia Clostridiales Syntrophomonadaceae sf_5 2456 granular sludge done R4bl4

Firmicutes Bacilli Bacillales Thermoactinomycetaceae sf_l 3301 Thermoactinom ces sp. str. 700375

Firmicutes Unclassified Unclassified Unclassified sf_8 546 Ferribacter thermoautotrophicus

Firmicutes Clostridia Clostridiales Unclassified sf_17 2324

Firmicutes Desulfotomaculum Unclassified Unclassified sf_l 2351 Desulfotomaculum thermobenzaicum str. DSM 6193

Firmicutes Desulfotomaculum Unclassified Unclassified sf_l 2359 UASB granular sludge done JP

Firmicutes Clostridia Unclassified Unclassified sf_3 2373

Firmicutes Symbiobacteria Symbiobacterales Unclassified sf_l 2388 G+C Gram-positive done YNPRH70A

Firmicutes Unclassified Unclassified Unclassified sf_8 2433 Ferribacter mermoautotrophicus str.

JW/JH-Fiji-2

Firmicutes Desulfbtomaculum Unclassified Unclassified sf_l 2443 Desulfbtomaculum thermoacetoxidans str. DSM

5813

Firmicutes Desulfotomaculum Unclassified Unclassified sf_l 2490 Desulfotomaculum solfataricum str. V21

Firmicutes Clostridia Unclassified Unclassified sf_4 2398 deep marine sediment done MB-C2-106

Firmicutes Symbiobacteria Symbiobacterales Unclassified sf_l 77 thermal soil done YNPFFP9

Firmicutes Clostridia Clostridiales Unclassified sf_17 926

Firmicutes Desulfbtomaculum Unclassified Unclassified sf_l 198 Pelotomaculum sp. str. JT

Firmicutes Catabacter Unclassified Unclassified sf 4 2716 termite gut homogenate done Rs-F76 bacterium

Firmicutes Clostridia Clostridiales Unclassified sf_17 3476

Firmicutes Bacilli Lactobacillales Unclassified sf_l 3289 Isobaculum melis CCUG 37660T

(continued)

TABLE 3. (CONTI UBD)

Phylum Class Order Family S-F* Taxon IE Re resentative species

Firmicutes Bacilli Lactobacillales Unclassified sf 1 3481

Firmicutes Clostridia Qostridiales Unclassified sf 17 4168

Firmicutes Catabacter Unclassified Unclassified sf 4 4503 termite gut homogenate clone Rs-H83 bacterium

Firmicutes Unclassified Unclassified Unclassified sf_8 4536 Mono Lake at depth 35m station 6 July 2000 clone ML635J-14 G+C

Firmicutes Qostridia Unclassified Unclassified sf 7 4216

Firmicutes Catabacter Unclassified Unclassified sf 1 4261 tennite gut homogenate clone Rs-G04 bacterium

Firmicutes Catabacter Unclassified Unclassified sf 1 4293 termite gut homogenate clone Rs-QOl bacterium

Firmicutes gut clone group Unclassified Unclassified sf 1 4298 human mouth clone P4PA_66

Firmicutes Clostridia Qostridiales Unclassified sf_17 4307

Firmicutes Catabacter Unclassified Unclassified sf 4 4526 TCEKxmtaminated site clone ccslm210

Firmicutes gut clone group Unclassified Unclassified sf 1 4616 rumen clone F23-C12

Fusobacteria Fusobacteria Fusobacterales Fusobacteriaceae sf 3 367 Leptotrichia amnionii str. AMN-1

Fusobacteria Fusobacteria Fusobacterales Fusobacteriaceae sf 3 558 Sneathia sanguinegens str. CCUG 41628T

Fusobacteria Fusobacteria Fusobacterales Fusobacteriaceae sf_l 488 Fusobacterium nucleatum subsp. vincentii str.

ATCC 49256

Gemmatimonadetes Unclassified Unclassified Unclassified sf 5 442 forest soil clone S0134

Gemmatimonadetes Unclassified Unclassified Unclassified sf_5 227 uranium mining waste pile clone JG37-AG-36

Gemmatimonadetes Unclassified Unclassified Unclassified sf_5 9464 lodgepole pine rhizosphere soil British Columbia

Ministry Forests Long-Term Soil Productivity

Gemmatimonadetes Unclassified Unclassified Unclassified sf 5 10112 forest soil done NOS7.157WL

Gemmatimonadetes Unclassified Unclassified Unclassified sf 5 317 penguin droppings sediments clone KD8-87

Gemmatimonadetes Unclassified Unclassified Unclassified sf_5 1127 uranium mining waste pile near

Johanngeorgenstadt soil clone JG37-AG-21

Gemmatimonadetes Unclassified Unclassified Unclassified sf 5 1565 uranium mining waste pile clone JG34-KF-418

Gemmatimonadetes Unclassified Unclassified Unclassified sf 5 2047 soil clone #0319-7G21

LD1PA group Unclassified Unclassified Unclassified sf_l 10118 anoxic marine sediment clone LD1-PA38

Lentisphaerae Unclassified Unclassified Unclassified sf 5 10027 Cytophaga sp. str. Dex80-43

Lentisphaerae Unclassified Unclassified Unclassified sf 5 10330 Mono lake clone ML635J-58

Lentisphaerae Unclassified Unclassified Unclassified sf 5 9704 Cytophaga sp. str. Dex80-64

marine group A mgA-2 Unclassified Unclassified sf 1 6344 bacterioplankton clone ZA3648c

marine group A mgA-1 Unclassified Unclassified sf 1 6408 Sargasso Sea

marine group A mgA-1 Unclassified Unclassified sf 1 6454 marine clone SAR406

Natronoanaerobium Unclassified Unclassified Unclassified sf 1 769 fjord ikaite column clone un-c23

Natronoanaerobium Unclassified Unclassified Unclassified sf_l 2437 Mono Lake at depth 23m station 6 July 2000 clone ML623J-19

Natronoanaerobium Unclassified Unclassified Unclassified sf 1 3570 Bacillus sp. clone ML1228J-1

Natronoanaerobium Unclassified Unclassified Unclassified sf_l 3745 Mono Lake at depth 35m station 6 July 2000 clone ML635J-45

Natronoanaerobium Unclassified Unclassified Unclassified sf_l 4377 Mono Lake at depth 35m station 6 July 2000 clone ML635J-65 G+C

NC10 NClO-1 Unclassified Unclassified sf 1 452 vadose clone 5G01

NC10 NClO-1 Unclassified Unclassified sf 1 536 uranium mill tailings clone GuBH2-AD-8

NC10 NClO-2 Unclassified Unclassified sf 1 10254 uranium mill tailings soil sample clone

Sh765B-TzT-35

Nitrospira Nitrospira Nitrospirales Nitrospiraceae sf_l 984 uranium mining waste pile done JG37-AG-131 sp.

Nitrospira Nitrospira Nitrospirales Nitrospiraceae sf_2 542 forested wetland done FW19

Nitrospira Nitrospira Nitrospirales Nitrospiraceae sf_2 544 forested wetland done FW5

Nitrospira Nitrospira Nitrospirales Nitrospiraceae sf_2 697 forested wetland done FW118

OP10 CH21 cluster Unclassified Unclassified sf_l 326 geothermal done ST01-SN3H

OP10 Unclassified Unclassified Unclassified sf_4 484 forested wetland done FW68

OP10 CH21 cluster Unclassified Unclassified sf_l 514 sludge done SBRA136

OP10 Unclassified Unclassified Unclassified sf_5 9782 Rocky Mountain alpine soil done Sla-IH

OP3 Unclassified Unclassified Unclassified sf_4 628 CB<ontaminated groundwater done GOUTB15

OP3 Unclassified Unclassified Unclassified sf_2 349 soil done PBS-25

OP9/JS1 OP9 Unclassified Unclassified sf_l 726 hot spring done OPB72

OP9/JS1 OP9 Unclassified Unclassified sf_l 969 DCP½ecWorinating consortium done SHA-1 phylum_tax classjax orderjax family_tax subfamily otu_id rep_prokMSAname

Planctomycetes Planctomycetacia Planctomycetales Anammoxales sf_2 4683 anoxic basin clone CY0ARA028B09

Planctomycetes Planctomycetada Planctomycetales Anammoxales sf_4 4694 USA: Colorado Fort coUins Horsetooth Reservoir done HT2F11

Planctomycetes Planctomycetacia Planctomycetales Anammoxales sf_4 9662 Great Artesian Basin done B83

Planctomycetes Planctomycetacia Planctomycetales Pirellulae sf_3 4670

Planctomycetes Planctomycetacia Planctomycetales Pirellulae sf_3 4677 aerobic basin done CY0ARA032A03

Planctomycetes Planctomycetacia Planctomycetales Planctomycetaceae sf_3 4652 anoxic basin done CY0ARA028C04

Planctomycetes Planctomycetacia Planctomycetales Planctomycetaceae sf_3 4948 anoxic basin done CY0ARA027D01

Proteobacteria Alphaproteobacteria Acetobacterales Acetobacteraceae sf_l 7529 Gluconacetobacter europaeus str. ΖΓΜ B028 V3

Proteobacteria Gammaproteobacteria Acidithiobacillales Addithiobacillaceae sf_l 8320 add mine drainage done BA11

Proteobacteria Gammaproteobacteria Addithiobacillales Addithiobadllaceae sf_l 8552 Acidithiobacillus ferrooxidans str. D2

Proteobacteria Gammaproteobacteria Acidithiobacillales Addithiobacillaceae sf_l 9224 Acidithiobacillus albertensis str. DSM 14366

Proteobacteria Gammaproteobacteria Addithiobacillales Addithiobacillaceae sf_l 9497 Acidithiobacillus ferrooxidans str. ATCC 19859

Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae sf_l 9294 Arctic deep sea Isolation common

chemoorganotrophic oxygen-respiring polar current d 1210

Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae sf_l 8340 Aeromonas ichthiosmia

Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae sf_l 8364 Aeromonas a osaccharophUa str. CECT 4199

Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae sf_l 8621 Aeromonas sp. PAR2A

Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae sf_l 9000 Aeromonas culicicola str. MTCC 3249

Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae sf_l 9026 Haemophilus piscium str. NCI B 1952

Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae sf_l 9440 Aeromonas sobria str. NCIMB 12065

Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae sf_l 9494 Aeromonas molluscorum str. 849T

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 7737 atrazine-catabolizing microbial presence

methanol done RA30+06A

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 7768 swine intestine done p-861-a5

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 7788 atrazine-catabolizing microbial absence

methanol done KRA30-58

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 7838 Alcaligenes defragrans str. PD-19

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 7902 Alcaligenes faecalis str. M3A

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 7932 Adiromobacter subsp. denitrificans str. DSM

30026 (T)

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 7984 Waste-gas biofilter done BIfdii38

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 7992 Alcaligenes faecalis 5659-H

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 8062 BracMelh oedipodis str. LMG 1945 R8846

{continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family Taxon /D ^B Representative species

Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae sf_l 8094 A aligenes sp. str. VKM B-2263 dcm6 Proteobacteria Gammaproteobacteria Oceartospirillales Alcanivoraceae sf_l 8335 Alcanworax sp. str. K3-3 ( BIC 4323) Proteobacteria Gammaproteobacteria Oceanospirillales Alcanivoraceae sf_l 9658 Alcanworax sp. str. Hawl

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9035 Microbulbifer sp. str. JAMB-A94

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8348 Arctic sea ice ARK10038

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8484 Alteromonadaceae isolate str. LA50

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8503 Arctic sea ice ARK10244

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8578 Marinobacter lipolyticus str. SM-19

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8594 Marinobacter sp. str. SBS

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9239 Arctic sea ice ARK10228

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8196

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8222

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8600 ColweWa piezophua str. Y223G

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8753 Idiomarina loihiensis str. GSP37

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8174 attached marine recovered surface clone 17 proteobacterium

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8318 Aestuariibacter salexigens str. JC2042

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8374 Agarworans albus str. MKT 89

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8533

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8695 Arctic pack ice; northern Fram Strait; 80 31.1 N;

CO 01 deg 59.7 min E clone ARKIA-34O Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8863 Alteromonas marina str. SW-47

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8970 Arctic seawater isolate str. R9879

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8978 Arctic sea ice ARK10108

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9230 Antarctic pack ice Lasarev Sea Southern Ocean clone ANTXI/4_14-62 sea

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9236 attached marine recovered surface clone 18 proteobacterium

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sfJL 9288 Alteromonas stettipolaris str. LMG 21861 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9292

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9501 sea water isolate str. BP-PH

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9562 Alteromonadaceae clone PH-B55N

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8172 Pseudodteromonas sp. str. Bdeep-1

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8336 Alteromonas sp. str. MS23

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8580 Arctic seawater isolate str. R7076

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8932 Pseudodteromonas antarctica str. N-l

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 8975 Alteromonas sp. str. IBH P1M3

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9058 Pseudodteromonas carrageenovora str. ATCC

12662T

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9111 Pseudodteromonas sp. str. E36

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9143 Pseudodteromonas agarivorans str. KMM 255 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9205 marine clone Arctic96B-17

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9218 Pseudodteromonas hdoplanktis str. ATCC 14393 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9324 Pseudodteromonas ruthenica str. KMM300 Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf_l 9386 Alteromonas sp. str. NIBH P2M11

Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf_l 9640 exposed to diatom detritus isolate str. Tw-10

Tw-10

Proteobacteria Ganunaproteobacteria Alteromonadales Alteromonadaceae sf 1 8643 Pseudodteromonas porphyrae str. S2-65 Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf 1 9369 Pseudodteromonas luteamolacea str. NCTMB 1893T Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf 1 9222 Shewanella hanedai str. CUP 103207T

Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf 1 9384 Moritella viscosa str. NVI 88/478T

Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf 1 8916 ShewaneUa algae str. 43940

Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf 1 9067 ShewaneUa dgae str. ACM 4733

Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf 1 9416 marine isolate str. R8

Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf 1 9586 Shewanella gaetbuli str. TF-27

Proteobacteria Garnmaproteobacteria Alteromonadales Alteromonadaceae sf 1 8579 Psychromonas profunda str. 2825

Proteobacteria Alphaproteobacteria Rickettsiales Anaplasmataceae sf 3 6628 Vfolbachia pipientis

Proteobacteria Alphaproteobacteria Rickettsiales Anaplasmataceae sf 3 6648 Wolbachia sp

Proteobacteria Alphaproteobacteria Rickettsiales Anaplasmataceae sf 3 6803 Wolbachia sp. Dleml6SWol

Proteobacteria Alphaproteobacteria Rickettsiales Anaplasmataceae sf 3 6908 Rhmocyllus amicus endosymbiont

Proteobacteria Alphaproteobacteria Rickettsiales Anaplasmataceae sf 3 7481 Wolbachia pipientis

Proteobacteria Alphaproteobacteria Rhizobiales Bartonellaceae sf 1 7056 Bartonella schoenbuchensis str. Rl

Proteobacteria Alphaproteobacteria Rhizobiales Bartonellaceae sf_l 7384 aortic heart valve patient with endocarditis clone v9

Proteobacteria Alphaproteobacteria Rhizobiales Bartonellaceae sf 1 7415 Bartonella quintana str. Toulouse

Proteobacteria Alphaproteobacteria Rhizobiales Bartonellaceae sf 1 7634 Bartonella henselae str. Houston-1

Proteobacteria Deltaproteobacteria Bdellovibrionales Bdellovibrionaceae sf_l 10010 uranium mining waste pile clone JG37-AG-139 proteobacterium

Proteobacteria Alphaproteobacteria Bradyrhizobiales Beijerinck/Rhodoplan/ sf_3 7401 Scrippsiella trochoidea NEPCC 15

CD Methylocyst

O Proteobacteria Alphaproteobacteria Bradyrhizobiales Beijerinck/Rhodoplan/ sf_3 6651 Beijerinckia indica

Methylocyst

Proteobacteria Alphaproteobacteria Bradyrhizobiales Beijerinck/Rhodoplan/ sf_3 7275 Mammoth cave clone CCU18

Methylocyst

Proteobacteria Alphaproteobacteria Bradyrhizobiales Beijerinck/Rhodoplan/ sf_3 7219 Methylosinus sporium

Methylocyst

Proteobacteria Alphaproteobacteria Bradyrhizobiales Beijerinck/Rhodoplan/ sf_3 7640 Methylosinus trichosporium

Methylocyst

Proteobacteria Alphaproteobacteria Bradyrhizobiales Beijerinck/Rhodoplan/ sf_3 6762 acidic forest soil clone UP8

Methylocyst

Proteobacteria Alphaproteobacteria Bradyrhizobiales Beijerinck/Rhodoplan/ sf_3 7153 Methylocella tundrae str. Yl

Methylocyst

Proteobacteria Alphaproteobacteria Rhizobiales Bradyrhizobiaceae sf 1 7029

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 7403 Oligotropha carboxidovorans str. S23

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 6927 Nitrobacter hamburgensis str. X14

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 6768 Rhodopseudamonas pdustris str. GH

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 6799 Rhodopseudamonas pdustris str. ATCC 17001 Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 7316

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 7333 Aftpia genosp. 4 str. G3644

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 6941 Rhodopseudamonas rhenobacensts str. Klemme Kb Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 7087 Br dyrhizobium japonicum HA1

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 7398 Bradyrhizobium japonicum str. USDA 38 Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sU 6636 Bradyrhizobium elkanii str. USDA 76

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae sf 1 6867 heavy metal<ontaminated soil clone a!3131

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family Representative species'

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae Bradyrhizobium str. YB2

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae Afipia genosp. 2 str. G4438

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae ground water deep-well injection disposal site radioactive wastes Tomsk-7 clone S15A-MN96 proteobacterium

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae Afipia genosp. 10 str. G8996

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae Bradyrhizobium elk nii str. SEMIA 6028 Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae Bradyrhizobium sp. str. KKI14

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae Bradyrhizobium j ponicum SD5

Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae Bradyrhizobium japonicum str. LAM 12608 Proteobacteria Alphaproteobacteria Bradyrhizobiales Bradyrhizobiaceae temperate esruarine mud clone HC65 Proteobacteria Alphaproteobacteria Rhizobiales Brucellaceae Ochrobactrum anthropi str. ESC1

Proteobacteria Alphaproteobacteria Rhizobiales Brucellaceae Ochrobactrum gallmifaecis str. Iso 196

Proteobacteria Alphaproteobacteria Rhizobiales Brucellaceae

Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae penguin droppings sediments clone KDl-79 Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Burkholderia glathei str. ATCC 29195T Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Burkholderia hospita str. IMG 20598T

Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Burkholderia sp.

Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Burkholderia caribensis str. MWAP71

Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae Burkholderia caryophylli str. ATCC 25418 Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae

Proteobacteria Alphaproteobacteria Consistiales Caedibacteraceae acid mine drainage clone ASL45

Proteobacteria Alphaproteobacteria Consistiales Caedibacteraceae termite gut homogenate done Rs-B60

proteobacterium

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae deepest cold-seep area Japan Trench clone

JTB360 proteobacterium

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Riftia pachyptila's tube done R103-B70 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Arcobacter cryaerophUus

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae SulfurospirUlum deleyianum str. Spirillum 5175 Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter sp. str. N02B

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter gracilis

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter showae

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter subsp. fetus

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter helveticus

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Campylobacter showae str. IMG 12636 Proteobacteria Garnmaproteobacteria Cardiobacteriales Cardiobacteriaceae Cardiobacterium hominis

Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Asticcacaulis excentricus str. ATCC15261 Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Brevundimonas intermedia str. MBIC2712

ATCC15262

Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Brevundimonas vesiculitis str. IAM 12105T Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Brevundimonas diminuta str. DSM 1635 Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Brevundimonas diminuta str. IAM 12691T Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Brevundimonas bacteroides str. CB7

Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae Brevundimonas subwbrioides str. CB81

Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae sf 1 7436 Bretmndimonas sp. str. FWC40 Proteobacteria Gammaproteobacteria Chroma tiales Chromatiaceae sf 1 9048 Allochromatium sp. AT2202

Proteobacteria Gammaproteobacteria Chroma tiales Chroma tiaceae sf 1 8546 Thiocapsa litoralis

Proteobacteria Gammaproteobacteria Chroma tiales Chromatiaceae sf 1 8527

Proteobacteria Gammaproteobacteria Chromatiales Chroma tiaceae sf 1 8697 Thiococcus sp. AT2204

Proteobacteria Gammaproteobacteria Chromatiales Chromatiaceae sf 1 9054

Proteobacteria Gammaproteobacteria Chromatiales Chromatiaceae sf 1 9052

Proteobacteria Gammaproteobacteria Chromatiales Chromatiaceae sf 1 9356

Proteobacteria Gammaproteobacteria Chromatiales Chromatiaceae sf 1 9370 isolate str. HTB019

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 8112 Comamonas testosteroni str. SMCC B329 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf_l 7704 freshwater clone PRD01b009B

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7705 penguin droppings sediments clone KD4-7 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf_l 7801 Toolik Lake main station at 3 m depth clone

TLM05/TLMdggelO proteobacterium

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7829 Xylophilus ampelmus str. ATCC 33914 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7928 penguin droppings sediments clone D5- 3 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf_l 7941 MCB<ontaminated groundwater-treating reactor clone RB9C10

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7986 Arctic sea ice ARK10281

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 8138 Pseudomonas lanceolata str. ATCC 14669T Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf_l 8139 Delfti tsuruhatensis str. AD9

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7856 V riovorax paradoxus

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7964 napthalene-contarninated sediment clone 76 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7888 Hydrogenophaga flaoa str. DSM 619T

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7919 strain isolate str. rM4

r Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7987 Acidovor x sp. str. OS-6

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 8012 Acidovorax onjaci str. DSM 7481

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 8018 Acidovorax delafieldii str. ATCC 17505 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf_l 8021 Acidovorax jacilis str. CCUG 2113

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf_l 8022 Acidovorax avenae subsp. catfleyae str. NCPPB

961 subsp.

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 8031 strain isolate str. rJlO

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf_l 8046 Acidovorax defluvii str. BSB411

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 8152 nephridia Octolasion lacteum clone 012-2 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7807 Aquaspirillum metamorphum str. DSM 1837 Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7884 GermanyrElbe River clone Hb37

Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae sf 1 7965 Anoxobacteri m dechloraticum

Proteobacteria Gammaproteobacteria Legionellales Coxiellaceae sf 3 7893 agricultural soil clone SC-I-71

Proteobacteria Gammaproteobacteria Legionellales Coxiellaceae sf 3 8457 5' clone CHAB-XI-27

Proteobacteria Gammaproteobacteria Legionellales Coxiellaceae sf_3 9198 uranium mining waste pile clone F-JG30-B15

KF-JG30-B15

Proteobacteria Gammaproteobacteria Legionellales Coxiellaceae sf_3 8969 uranium mining waste pile soil sample clone

JG30-KF-C15 proteobacterium

Proteobacteria Gammaproteobacteria Legionellales Coxiellaceae sf 3 9444 forested wetland clone FW23

Proteobacteria Deltaproteobacteria Desulfbbacterales Desulfoarculaceae sf 2 10227 marine sediment clone Bolll

Proteobacteria Deltaproteobacteria Desulf bacterales Desulfobacteraceae sf_5 9666 marine sediment above hydrate ridge clone

Hyd89-13 proteobacterium

Proteobacteria Deltaproteobacteria Desulf bacterales Desulfobacteraceae sf_5 9875 hydrothermal sediment clone AF420354

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family S-F" Taxon I& Representative species

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae sf 5 9800 forested wetland clone FW57

Proteobacteria Deltaproteobacteria Desulfbbacterales Desulfobacteraceae sf 5 10268

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae sf_5 10046 Desulfobacterium cetonicum str. DSM 7267 oil recovery water

Proteobacteria Deltaproteobacteria Desulfbbacterales Desulfobacteraceae sf 5 10239

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae sf 5 10319 sulfate-reducing habitat clone SLM-CP-116

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae sf 5 10031 Antarctic sediment clone SB1_49

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae sf 5 10083 Desulfobacter curvatus str. DSM 3379

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae sf 5 9940 Antarctic sediment clone SB2J56

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobulbaceae sf 1 10047 epibiontic clone C11-D3

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobulbaceae sf_l 10187 Mono Lake at depth 23 m station 6 July 2000 clone ML623J-57 proteobacterium

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobulbaceae sf 1 9734 Riftia pachyptik's tube clone R103-B13

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobulbaceae sf 1 9739 gas hydrate clone Hyd89-51

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfohalobiaceae sf 1 9894 Oesuljbnauticus submarinus str. 6N

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfornicrobiaceae sf 1 10079 Desulfbmicrobium baculatum str.. DSM 1742

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae sf 1 10262 Desulfovibrio sp. str. Ac5.2

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae sf 1 10248 Desulfovibrio giganteus str. DSM 4370

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae sf_l 10016 termite gut homogenate clone Rs-N35

proteobacterium

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae sf_l 9826 termite gut homogenate clone Rs-M72

proteobacterium

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae sf_l 10071 Desulfovibrio desulfuricans

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae sf 1 10212

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfovibrionaceae sf_l 9709 termite gut homogenate clone Rs-N31

proteobacterium

Proteobacteria Deltaproteobacteria Desulfuromonadales Desulfuromonaceae sf_l 10020 uranium mill tailings soil sample clone GuBH2-

AG-114 proteobacterium

Proteobacteria Gammaproteobacteria Chromatiales Ectothiorhodospiraceae sf 1 9450 Halorhodospira neutrophila str. SG 3304

Proteobacteria Gammaproteobacteria Chromatiales Ectothiorhodospiraceae sf_l 9598 Mono Lake at depth 2 m station 6 July 2000 clone

ML602J-47 proteobacterium

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf 6 433 coal effluent wetland clone RCP2-6

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf 6 646 Opitutus sp. str. SA-9

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf 1 9309 Buchnera sp

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8742 USA:New York isolate str. KN4

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf 6 8783 Alterococcus agaroh/ticus str. ADT3; CCRC17102

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf 1 9135 intestine Zophobas mm clone

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9358 Salmonella subsp. enterica serovar Waycross str.

Swyl subsp.

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf 1 9496

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8886 Salmonella typhimurium LT2 str. SGSC1412

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf 1 8740 Erwinia chrysanthemi str. 573

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9651 Pectobacterium subsp. atrosepticum str. GSPB

i7in

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8379 Erwinia amylooora EA G-5

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9142 Erwinia amylooora str. DSM 30165

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9252 Pantoea cedenensis str. A34

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9345 Erwinia amylooora str. BC199(= Ea528) Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8554 Kluyvera ascorbata 69

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8885 MorganeUa morganii str. AP28

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9363 Citrobacter freundii str. CDC 621-64

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9594 MorganeUa morganii str. ATCC35200

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8758 Pectobacterium cypripedii str. ATCC 29267 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8282 Antonina pretiosa symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8693 Pantoea agglomerans str. A40

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8700 Baumannia cicadellinicola

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9302 Pantoea subsp. stewartii str. GSPB 2626 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8236 Vryburgia amaryllidis symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8504 Dysmicoccus neobreuipes symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8603 Melanococcus albizziae symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8607 Amonostherium lichtensioides symbiont Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8624 Erium globosum symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9290 Baumannia cicadellinicola

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9293 USA clone 14/7

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9420

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8934 Pectobacterium subsp. carotovorum str. E155 subsp.

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9266 Parasite BEV of E.variegatus

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8467 Serratia marcescens subsp. sakuensis str. KRED subsp.

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9348

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8505 Buttiauxella warmboldiae str. DSM 9404 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8528 Enterobacter cloacae Nr. 3

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8530 Enterobacteriaceae CFOlEnt-1

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8640

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8936 Klebsiella oxytoca str. ChDC OS31

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9060 Enterobacter ludwigii str. EN-119 = DSMZ 16688 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9274 Enterobacter sp. CC1

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9361 Enterobacter intermedins str. JCM1238

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae s l 9390 Enterobacter nimipressuralis str. LMG 10245-T Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8251 Nitrogen-fixing isolate str. CANP3

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8529 Raoultella plantkola 7

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8627 Australicoccus grem&eae symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8770

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8890 RaoulteUa planticola str. DR3

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8362 Klebsiella pneumoniae str. ASR1

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8510 Klebsiella pneumoniae str. DSM 30104

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8773

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8286 Cyphonococcus alpinus symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8711 Serratia odorifera str. DSM 4582

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8712 Serratia proteamaculans str. DSM 4543 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8739 Serratia entomoph a str. DSM 12358

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8892 Aranicola proteolyticus

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9151

(continued)

TABLE 3. (CO TINUBD)

Phylum Class Order Family Taxm Kr" Representative species?

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9417 Serrati fimticola str. DSM 4576

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8631 Planococcus ficus symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8283 Heteropsylla texana symbiont

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8173 Photorhabdus asymbiotica str. ATCC 43949 Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8225

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8642 Erwinia chrysanthemi str. 580

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9029 Photorhabdus asymbiotica subsp. australis str. MB Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8473 Hafnia lvei

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9265 Rahnelk aquatilis k 8

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9337 Rahnella genosp. 3 str. DSM 30078

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 8564 Rahnelk aquatilis str. ATCC 33989

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9157 Secondary symbiont type-U Acyrthosiphon pisum (rrs) clone 5B type-U

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 9262 Yersinia aldovae str. A125

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf_l 1206 Dermacentor variabilis symbiont

Proteobacteria Gammaproteobacteria Thiotrichales Francisellaceae sf_l 9554 Ti!apia parasite TPT-541

Proteobacteria Gammaproteobacteria Thiotrichales Francisellaceae sf_l 8949 Caedibacter taeniospiralis

Proteobacteria Deltaproteobacteria Desulfuromonadales Geobacteraceae sf_l 482 trid oroethene caitaminated site clone

FTLM205 proteobacterium

Proteobacteria Deltaproteobacteria Desulfuromonadales Geobacteraceae sf_l 10171

CO Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae sf_l 8514 Chromohalobacter israelensis str. ATCC 43985 T en Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae sf_l 8562 Halomonas sp. str. TNB L20

Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae sf_l 8576 Halomonas sp. o502

Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae sf_l 8598 Halomonas desiderata str. FB2

Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae sf_l 8854 Halomonas variabilis str. A T9112

Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae sf_l 9471 Boston Harbor surface water isolate str.

UMB18C UMB18C

Proteobacteria Gammaproteobacteria Oceanospirillales Halomonadaceae sf_l 9141 Halomonas sp. SKI

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10385

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10428 Flexispira rappini FH 9702248

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10430 Helicobacter heUmannii str. MM2

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10436 Helicobacter aurati str. ΜΓΓ 97-5075c

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10442 Helicobacter cetorum str. MIT 99-5656 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10444 Helicobacter suncus str. Kaz-2

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10448 Helicobacter felis str. Dog- Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10451 Helicobacter heUmannii str. C4S

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10454 Helicobacter pullorum str. NCTC 12826 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10462 Helicobacter rodentium str. ΜΓΓ 96-1312 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10518 Helicobacter pylori str. ATCC 49396T

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10520 Helicobacter sp. blood isolate 964

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10548 Helicobacter rappini W.Tee-Bat

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sL3 10552 Helicobacter winghamensis str. NLEP 97-1611 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10562 Helicobacter rappini W.Tee-Yu

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10425 Sulfurimonas autotrophica str. OK5

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10411 termite gut homogenate clone Rs-P71

proteobacterium

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10432 Riftia pachyptMs tube clone R76-B51

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10438 hydrocarbon seep clone GCA014

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10590 termite gut homogenate clone Rs-H40

proteobacterium

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10614 strain isolate str. BHI80-49

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10417 temperate estuarine mud clone KM61 Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10467

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf_3 10507 termite gut homogenate clone Rs-M59

proteobacterium

Proteobacteria Alphaproteobacteria Bradyrhizobiales Hyphomicrobiaceae sf 1 7646 Hyphomicrobium aestuarii str. DSM 1564 Proteobacteria Alphaproteobacteria Rhizobiales Hyphomicrobiaceae sf 1 7392

Proteobacteria Gammaproteobacteria Legionellales Legionellaceae sf_l 8865 Arctic pack ice; northern Pram Strait; 80 31.1 N;

01 deg 59.7 min E clone ARKCH2Br2-23

Proteobacteria Alphaproteobacteria Azospirillales Magnetospiiillaceae sf 1 6922 Dechlorospirittum sp. str. SN1

Proteobacteria Alphaproteobacteria Bradyrhizobiales Methylobacteriaceae sf 1 7585 Methylobacterium thiocyanatum str. ALL/SCN-P Proteobacteria Gammaproteobacteria Methylococcales Methylococcaceae sf 1 8243 isolate str. E

Proteobacteria Gammaproteobacteria Methylococcales Methylococcaceae sf 1 8821 Methylobacter psychrophilus str. Z-0021 Proteobacteria Gammaproteobacteria Methylococcales Methylococcaceae sf_l 9438 marine sediment above hydrate ridge clone

Hyd24-01 proteobacterium

Proteobacteria Betaproteobacteria Methylophilales Methylophilaceae sf_l 8137 freshwater clone PRDOlaOllB

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf_3 8366 Psychrobacter frigidicola str. DSM 12411 Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf_3 8604 MoraxeU oblonga str. LAM 14971

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf 3 8838 Psychrobacter psychrophilus CMS 28

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf 3 8727 A&anindiges hongkongensis str. HKU9

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf 3 9359 Acinetobacter junii str. S33

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf_3 9428 hydrocarbon-degrading consortium clone

AF2-1D

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf 3 9466 Acinetobacter landoii str. 4N13

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf 3 9641 Acinetobacter haemolyticus

Proteobacteria Deltaproteobacteria Myxococcales Myxococcaceae sf_l 10358 Myxococcus fulvus str. Mx f2

Proteobacteria Epsilonproteobacteria Nautiliales Nautiliaceae sf_l 10477 S17sBac5 complete clone

Proteobacteria Betaproteobacteria Neisseriales Neisseriaceae sf 1 7945 Aouaspiri um serpens str. LAM 13944

Proteobacteria Betaproteobacteria Neisseriales Neisseriaceae sf 1 7675 Neisseria sp. str. CCUG 46910

Proteobacteria Betaproteobacteria Neisseriales Neisseriaceae sf_l 7662 Mars Odyssey Orbiter and encapsulation facility clone T5-1 sp.

Proteobacteria Betaproteobacteria Nitrosomonadales Nitrosomonadaceae sf 1 7789

Proteobacteria Betaproteobacteria Nitrosomonadales Nitrosomonadaceae sf 1 7976 Nitrosomonas sp. str. Nm86

Proteobacteria Betaproteobacteria Nitrosomonadales Nitrosomonadaceae sf 1 7770 Nitrosomonas europaea str. ATCC 19718 Proteobacteria Betaproteobacteria Nitrosomonadales Nitrosomonadaceae sf 1 8145 Nitrosomonas eutropha str. Nm57

Proteobacteria Deltaproteobacteria Desulfobacterales Nitrospinaceae sL2 594 uranium mining mill tailing clone GR-296.II.52

GR-296.I.52

Proteobacteria Gammaproteobacteria Oceanospirillales Oceanospirillaceae sf_l 9351 bacterioplankton clone ZA2333c

Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae sf_l 7743 Herbaspirillum sp. str. NAH4

Proteobacteria Betaproteobacteria Burkholderiales Qxalobacteraceae sf 1 7843 Massilia timonae timone

Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae sf 1 7845 Diaphorina citri symbiont

Proteobacteria Betaproteobacteria Burkholderiales Qxalobacteraceae sf 1 7866 Paucimonas lemoignei str. ATCC 17989T Proteobacteria Betaproteobacteria Burkholderiales Qxalobacteraceae sf 1 7878 rtapthalene-«mtarninated sediment clone 29

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family Taxon IL Representative species

Proteobacteria Betaproteobacteria Burkholderiales Qxalobacteraceae sf_l 7921 Collimonas fungivorans str. Ter331

Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae sf_l 7968 Oxalobacter formigenes str. OXB ovinen rumen Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae sf_l 8013 isolate str. A1020

Proteobacteria Betaproteobacteria Burkholderiales Qxalobacteraceae sf_l 8032 Aquaspirillum arcticum str. IAM 14963 Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae sf_l 8034 Janthinobacterium ag ricidamnosum str. Wlr3T Proteobacteria Betaproteobacteria Burkholderiales Oxalobacteraceae sf_l 8058 Herbaspirillum seropedicae str. DSM 6445 ATCC

35892

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 9360 Pasteurella multodda subsp. gallidda str. MCCM

00021 subsp.

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 9349 Pasteurella sp. str. 91985

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8195 Haemophilus influenzae str. R2866

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8555 Haemophilus influenzae str. M9741

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 9213 Haemophilus quentini str. MCCM 02026 Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 9477 Haemophilus influenzae str. Ml 1105

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8228 Actinobatillus indolicus str. H1419

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8861 Haemophilus parasuis 427

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8614 Acidithiobactllus thiooxidans str. KCTC 8928P Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8952 Actinobacillus lignieresii

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 9263 Actinobacillus capsulatus

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8876 Mannheimk sp. R19.2 str. R19.2; CCUG 38463

R19.2

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 9237

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8409 human colonic mucosal biopsy done ABLCfl Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8432

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 8848 str. 86355

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 9533 Haemophilus segnis str. MCCM 00337

Proteobacteria Gammaproteobacteria Pasteurellales Pasteurellaceae sf_l 9628 Histophilus somni str. CCUG 12839

Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 6857 Mesorhizobium mediterraneum str. PECA20 Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 6692 Phyllobacterium trifolii str. PETP02

Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 6916 lake microbial mat isolate str. R-9219 Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 6966 Mesorhizobium tianshanense str. -IBS; USDA 3592 Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 7009

Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 7216 Ahrensia elensis str. IAM12618

Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 7379 Phyllobacterium myrsinacearum HM35

Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 7381 Aminobacter aminovorans str. DSM7048T Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 7497 Pseudaminobacter salicylatoxidans str. KTCOOl Proteobacteria Alphaproteobacteria Rhizobiales Phyllobacteriaceae sf_l 7300 marine isolate JP57

Proteobacteria Gammaproteobacteria Thiotrichales Pisdrickettsiaceae sf_3 8664 Thiomkrospira sp. str. Milos-T2

Proteobacteria Gammaproteobacteria Thiotrichales Pisc ckettsiaceae sf_3 9027 Thiomkrospira crunogena str. XCI _^ -2

Proteobacteria Gammaproteobacteria Thiotrichales Pisdrickettsiaceae sf_3 9557 Riftia pachyptila's tube done R76-B23 Proteobacteria Gammaproteobacteria Thiotrichales Pisdrickettsiaceae sf_3 9291 Methylophaga alcalica str. M39

Proteobacteria Gammaproteobacteria Thiotrichales Pisdrickettsiaceae sf_3 9392 Methylophaga sp. str. V4.ME.29=MM_2343 Proteobacteria Deltaproteobacteria Myxococcales Polyangjaceae sf_3 10249 soil sample uranium mining waste pile near town Johanngeorgenstadt done JG36-TzT-168 proteobacterium

Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf 3 10298 marine tidal mat clone BTM36 Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf 3 10353 sludge clone A9

Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf 3 9671 hydrothermal sediment clone AF420357 Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf_3 9735 uranium mining waste pile clone JG37-AG-15 proteobacterium

Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf 3 9755 bacterioplankton clone ZA3704c

Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf_3 9874 uranium mining waste pile clone JG34-KF-243 proteobacterium

Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf 3 9900 bioreactor clone mlel-27

Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf_3 10082 uranium mining waste pile clone JG37-AG-33 proteobacterium

Proteobacteria Deltaproteobacteria Myxococcales Polyangiaceae sf 4 9733 bacterioplankton clone ZA3735c

Proteobacteria Betaproteobacteria Procabacteriales Procabacteriaceae sf 1 8136 Acanthamoeba sp. UWC6 symbiont

Proteobacteria Gammaproteobacteria Alteromonadales Pseudoalteromonadaceae sf 1 9627 Pseudoalteromonas sp

Proteobacteria Gammaproteobacteria Alteromortadales Pseudoalteromonadaceae sf 1 9339 Pseudoalteromonas sp. str. 05

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8813 L rodus pedicellatus symbiont

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9300 Lyrodus pedicellatus symbiont

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8487

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8508 Pseudomonas citronellolis str. TERTDB26 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8691 Pseudomonas aeruginosa str. PAOl

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 8754 Pseudomonas sp. str. P400Y-1

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9002 Paederus fuscipes endosymbiont

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9056 Pseudomonas aeruginosa str. #47

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9588 Pseudomonas citronellolis str. TER1DB18

CD Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8288

00 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8777 Pseudomonas sp. str. KNA6-5

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8852 Pseudomonas stuizeri str. KC

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9068 . Pseudomonas stuizeri str. A1501

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 9228 Pseudomonas stutzeri HY-105

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9295

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 8344 Anabaena circinalis AWQC118C isolate str.

UNSW3

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 8553 Pseudomonas fulva str. IAM 1587

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8725 Pseudomonas sp. str. 2N1-1

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8850 Agrobacterium agile str. IAM12615

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9238

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9005 Pseudomonas sp. str. KY

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9613 Pseudomonas fiaoescens str. B62

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 8474 ground water deep-well injection disposal site radioactive wastes Tomsk-7 clone S15A-MNi proteobacterium

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8513 Pseudomonas monteUii str. CEP 104883 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 9049 uranium mining mill tailing clone GR-Sh2-34

GR-Sh2-34

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9219 Pseudomonas cf. monteUii 9

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9343 Cellvibrio subsp. mixtus str. ACM 2601 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9469 cf. Pseudomonas sp. clone Llangefni 52 Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9493 Pseudomonas sp. str. dcm7B

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family S-F* Taxon Representative species'

Proteobacteria Gammaproteobacteria Pseudomoruadales Pseudomonadaceae sf_l 8209 uranium mining waste pile clone JG37-AG-122 proteobacterium

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 8433 Pseudomonas syrmgae pv. broussonetiae str. KOZ

8101 pv.

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8635

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8853 Pseudomonas cichorii str. ATCC 10857T

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 9028 Pseudomonas koreensis str. Ps 9-14

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9240 . Pseudomonas fluorescens str. CHA0

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9267 Pseudomonas syringae pv. theae str. PTl

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9310 Pseudomonas sp. str. AC-167

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8338 Pseudomonas synxantha str. DSM 13080 G

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 8561 Pseudomonas sp. B65

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 8601 Pseudomonas margmalis str. ATCC 10844T

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8687 Pseudomonas putida str. ATCC 17472

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8708

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9175 Pseudomonas extremorientalis str. KMM3447

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 9221 Pseudomonas fulgida str. DSM 14938 = LMG 2146

P 515/12

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sU 9243 Pseudomonas tolaasii str. LMG 2342T 0

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9366 Arctic seawater isolate str. R7366

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8755 Pseudomonas sp. SK-1-3-1

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9172 Pseudomonas psychrophUa str. E-3

Proteobacteria Betaproteobacteria Burkholderiales Ralstoniaceae sf 1 7823 Wautersia bas ensis str. DSM 11853

Proteobacteria Betaproteobacteria Burkholderiales Ralstoniaceae sf 1 8110 Wautersia paucula str. LMG 3413

Proteobacteria Betaproteobacteria Burkholderiales Ralstoniaceae sf 1 8128 Cupriavidus necator

Proteobacteria Betaproteobacteria Burkholderiales Ralstoniaceae sf 1 7761 Ralstonia detusculanense str. APF11

Proteobacteria Betaproteobacteria Burkholderiales Ralstoniaceae sf 1 7778 Ralstonia insidiosa str. CCUG 46388

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 7051 Mycoplana dimorpha str. IAM 13154

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 6683 Sinorhizobiumfredii str. ATCC35423

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 6725 Sinorhizobium melQoti str. 1021

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 6972 Ensifer adhaerens str. LMG 20582

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf_l 6974 India: Himalayas Kaza Spiti Valley Cold Desert isolate str. Kaza-35 Kaza-35

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 6770 Rhizobium tropin str. LMG 9517

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 6871 Rkizobium mongolense str. USDA 1832

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf_l 7135 Rhizobium gallicum str. FL27

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf_l 7568 Rhizobium etli str. USDA 2667 ATCC 14483

SEMIA 043

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 6798 Agrobacterium tumefaciens TG14

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 6804 Rhizobium sp. str. SH19312

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 6964 Agrobacterium tumefaciens str. C58 Cereon

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf_l 7334 Agrobacterium tumefaciens C4

Proteobacteria Alphaproteobacteria Rhizobiales Rhizobiaceae sf 1 7041 Rhizobium huautlense str. S020

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf 1 6701 Roseobacter clone NACll-3

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf 1 6980 Loktanella vestfbldensis str. LMG 22003

Proteobacteria Alphaproteobacteria Khodobacterales Rhodobacteraceae sf_l 7433 Scrippsie trochoidea NEPCC 15

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf_l 7453 Sulfitobacter sp. BIO-11

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf_l 6888 hydrothermal vent strain str. TB66

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf_l 7026 Leisingera methylohalidivorans str. MB2 Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf_l 7263

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf_l 7040 Paracoccus alcaliphilus str. JCM 7364

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf_l 7508 lichen-dominated Antarctic cryptoendolithic community done FBP492 proteobacterium

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf_l 6991 Rhodobacter sphaeroides str. 2.4.1

Proteobacteria Alphaproteobacteria Rhodobacterales Rhodobacteraceae sf_l 7084 Scrippsklla trochoidea NEPCC 15

Proteobacteria Betaproteobacteria Rhodocy dales Rhodocyclaceae sf 1 7800 sample taken upstream landfill clone BVC77 landfill

Proteobacteria Betaproteobacteria Rhodoc dales Rhodocyclaceae sf_l 7817 TCE-contaminated site done ccs265

Proteobacteria Betaproteobacteria Rhodocy dales Rhodocyclaceae sf_l 7956

Proteobacteria Betaproteobacteria Rhodocy dales Rhodocyclaceae sf_l 8127 Zoogbea resintphUa str. PTV-3A2y

Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae sf_l 8131

Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae sf_l 7907 Thauera aromatica str. LG356

Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae sf_l 7925 Thauera selermtis str. ATCC 55363T

Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae sf_l 8156 industrial-phenol-degrading community done

MM1 sp.

Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae sf_l 7824 termite gut homogenate done Rs-B77

proteobacterium

Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae sf_l 7762 Elbe River snow isolate Isol8 Isol8_1411 Proteobacteria Alphaproteobacteria Rickettsiales Rickettsiaceae sf_l 7556 Rickettsia bellii str. strains 369-C and G2D42 Proteobacteria Gammaproteobacteria Oceanospirillales SaccharospiriUaceae sf_l 8889 hypersaline Mono Lake done MLllOJ-5 Proteobacteria Alphaproteobacteria Consistiales SARll sf_2 7043 marine done Arctic95D-8

Proteobacteria Gammaproteobacteria Alteromonadales Shewanellaceae sf_l 8581 Shewanella benthica str. DB21MT-2

Proteobacteria Gammaproteobacteria Alteromonadales Shewanellaceae sf_l 8641 Moritella b ssi str. 2693

Proteobacteria Gammaproteobacteria Alteromonadales Shewanellaceae sf_l 9081 Shewanella sp. str. MTW-1

Proteobacteria Gammaproteobacteria Alteromonadales Shewanellaceae sf_l 8662

Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 7440 Sphingobium chungbukense str. DJ77

Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 7528 Sphingobium yanoikuyae str. G J9882 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 7548 Afipia genosp. 13 str. G8991

Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 6650 Sphingomonas phyllosphaerae str. FA1

Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 7016 Sphingomonas sp. str. SAFR-027

Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 7535 Sphingomonas paucimob is str. GEFU2395 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_15 7035 Sphingomonas asaccharolytica str. IFO 10564-T Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 7215 travertine hot spring done SM2B06

Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 6663 Sphmgopyxis flavimaris str. SW-151

Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 7100 Novosphingobium capsulatum str. G J11526 Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf_l 7036 Lutibacterium anuloederans str. LC8

Proteobacteria Gammaproteobacteria Aeromonadales Suainivibrionaceae sf_l 8822 Anaerobiospirillum sp. str. 3J102

Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophaceae sf_3 10067 benzoate-degrading consortium done BA044 Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sf_l 9864 uranium mining waste pile done JG37-AG-133 proteobacterium

Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sf_l 10013 hydrothermal sediment done AF420341 Proteobacteria .Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sf_l 10021 uranium mill tailings soil sample done Sh765B-

TzT-29 proteobacterium

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family S-P Taxon ID ^b Representative species

Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sf_l 9731 uranium mining waste pile clone JG37-AG-90 proteobacterium

Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sf_l 9845 uranium mining waste pile clone JG37-AG-128 proteobacterium

Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sf 1 10184 granular sludge clone Rlp32

Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sU 10221 granular sludge clone R3p4

Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sf 1 10294 Oesulfacinum hydrothermak str. MT-96

Proteobacteria Deltaproteobacteria Syntrophobacterales Syntrophobacteraceae sf_l 9661 DCP-dechlorinating consortium clone SHD-1

Proteobacteria Gammaproteobacteria Thiotrichales Thiotrichaceae sf 3 8321 Wadden Sea sediment clone Dangast A9

Proteobacteria Garnrnaproteobacteria Thiotrichales Thiotrichaceae sf_3 8741 marine sediment clone Limfjorden L10

Proteobacteria Gammaproteobacteria Thiotrichales Thiotrichaceae sf 3 8752 Beggiatoa sp. str. MS-81-lc

Proteobacteria Gammaproteobacteria Thiotrichales Thiotrichaceae sf_3 9015 Beggiatoa alba str. B18LD; ATCC 33555

Proteobacteria Gammaproteobacteria Thiotrichales Thiotrichaceae sf 3 9321 marine sediment clone Tokyo Bay D

Proteobacteria Gammaproteobacteria Thiotrichales Thiotrichaceae sf 3 8703 Beggiatoa sp. str. AA5A

Proteobacteria Deltaproteobacteria Desulfbbacterales Unclassified sf 3 468 marine sediment clone Sva0515

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf 6 7377 Rocky Mountain alpine soil clone W2b-8C

Proteobacteria Alphaproteobacteria Verorhodospirilla Unclassified sf 1 7109 diesel-polluted Bohai Gulf isolate str. M-5 M-5

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf_6 7340 uranium mining waste pile soil sample clone

JG30-KF-AS50

Proteobacteria Alphaproteobacteria Azospirillales Unclassified sf 1 7400 sphagnum peat bog clone K-5b5

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf 6 6694 forested wetland clone RCP2-92

Proteobacteria Alphaproteobacteria Azospirillales Unclassified sf_l 6732 Anabaem circinalis AWQC118C isolate str.

UNSW7

Proteobacteria Alphaproteobacteria Acetobacterales Unclassified sf 1 7028

Proteobacteria Alphaproteobacteria EDin314/wr0007 Unclassified sf_l 7123 uranium mining waste pile near

Johaimgeorgenstadt soil clone JG37-AG-102

Proteobacteria Alphaproteobacteria EUin314/wr0007 Unclassified sf 1 7222 Great Artesian Basin clone B79

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf 6 7575

Proteobacteria Alphaproteobacteria Khizobiales Unclassified sf 1 6726

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf 6 6920 Pseudovibrio denitrificans str. DN34

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf 6 6954

Proteobacteria Alphaproteobacteria Ellin329/Rizl046 Unclassified sf_l 6945 Rhizobiales str. A48

Proteobacteria Alphaproteobacteria Bradyrhizobiales Unclassified sf 1 7067 Blastoch!oris sulfoviridis str. GN1

Proteobacteria Alphaproteobacteria Bradyrhizobiales Unclassified sf 1 7264 Bosea thiooxidans TJ1

Proteobacteria Alphaproteobacteria Rhizobiales Unclassified sf 1 7339

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf 6 6898 heavy metal<ontaminated soil clone al3113

Proteobacteria Alphaproteobacteria Bradyrhizobiales Unclassified sf 1 7199 uranium mill tailings done Gitt-KF-194

Proteobacteria Alphaproteobacteria Khizobiales Unclassified sf 1 6899

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf_6 6665 hydrocarbon-degrading consortium clone

4-Org2-22

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf_6 7312

Proteobacteria Alphaproteobacteria Rhizobiales Unclassified sf 1 6789 Shinella zoogloeoides str. ATCC 19623

Proteobacteria Alphaproteobacteria Unclassified Unclassified sU 6697 termite gut homogenate clone Rs-D84

proteobacterium

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf _2 7188 termite gut homogenate clone Rs-B50

proteobacterium

Proteobacteria Alphaproteobacteria Consistiales Unclassified sf_4 7105 Mariana trough hydrothermal vent water

0.2micro-m filterable fraction clone MT-NB25

Proteobacteria Alphaproteobacteria Rhodobacterales Unclassified sf_5 7471 sponge clone TK03

Proteobacteria Alphaproteobacteria Consistiales Unclassified sf_5 6735 Candidatus Pelagibacter ubique str. HTCC1002 Proteobacteria Unclassified Unclassified Unclassified s£_20 6763

Proteobacteria Alphaproteobacteria Rickettsiales Unclassified sf_2 6639

Proteobacteria Alphaproteobacteria Rickettsiales Unclassified sf_l 7156 termite gut homogenate clone Rs-M62

proteobacterium

Proteobacteria Deltaproteobacteria AMD clone group Unclassified sf_l 6830 coal effluent wetland clone RCP124

Proteobacteria Alphaproteobacteria Sphingomonadales Unclassified sf_l 6653 Kaistobacter koreensis str. PB229

Proteobacteria Deltaproteobacteria Bdellovibrionales Unclassified sf_l 7382 marine clone Arctic95C-5

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf_6 6987

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf_6 7572

Proteobacteria Betaproteobacteria Burkholderiales Unclassified sf_l 8035

Proteobacteria Betaproteobacteria MND1 clone group Unclassified sf_l 7808 Mammoth cave clone CCU25

Proteobacteria Betaproteobacteria Unclassified Unclassified sf_3 8007

Proteobacteria Betaproteobacteria Unclassified Unclassified sf_3 8036 Uranium mill tailings soil sample clone Sh765B-

TzT-132 proteobacterium

Proteobacteria Betaproteobacteria Unclassified Unclassified sf_3 7974

Proteobacteria Betaproteobacteria Unclassified Unclassified sf_3 8114

Proteobacteria Betaproteobacteria MND1 clone group Unclassified sf_l 8023 ferromanganous micronodule clone MND1 o Proteobacteria Betaproteobacteria Unclassified Unclassified sf_3 8045

IS) Proteobacteria Betaproteobacteria M D1 clone group Unclassified sf_l 7818 soil sample uranium mining waste pile near town Johanngeorgenstadt clone JG36-TzT-215 proteobacterium

Proteobacteria Betaproteobacteria Neisseriales Unclassified sf_l 8037 Chitinimonas tarwanensis str. cf

Proteobacteria Betaproteobacteria Unclassified Unclassified sf_3 7997

Proteobacteria Garnmaproteobacteria uranium waste clones Unclassified sf_l 8747 uranium waste soil clone JG30-KF-CM35 Proteobacteria Gammaproteobacteria GAO cluster Unclassified sf_l 9059 activated sludge clone SBRH10

Proteobacteria Garnmaproteobacteria aquatic clone group Unclassified sf_l 9246 Mammoth Cave sediment clone CCD24 Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 9498

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 9568 forested wetland clone RCP2-96

Proteobacteria Gammaproteobacteria Chromatiales Unclassified sf_l 9282

Proteobacteria Gammaproteobacteria Legionellales Unclassified sf_l 9418 uranium mining waste pile clone JG37-AG-14 proteobacterium

Proteobacteria Deltaproteobacteria EB1021 group Unclassified sf_4 8169 forested wetland clone RCP2-54

Proteobacteria Gammaproteobacteria Symbionts Unclassified sf_l 8403 Selenate-reducing isolate str. KE40H1 Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8488

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8646

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8676

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8926 inactive deep-sea hydrothermal vent chimneys clone IheB2-13

Proteobacteria Gammaproteobacteria aquatic clone group Unclassified sf_l 8957 marine clone Arctic97C-5

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 9105

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 9124 lOe-6 dilution marine samples Weser estuary clone DC8-80-1 proteobacterium

(continued)

TABLE 3. (CONTT WBD)

Phylum Class Order Family S-F" Taxon !D ^b Representative species''

Proteobacteria Gammaproteobacteria Symbionts Unclassified sf_l 9128 Lucina nassula gill symbiont

Proteobacteria Garnmaproteobacteria Unclassified Unclassified sf_3 9394

Proteobacteria Gammaproteobacteria Symbionts Unclassified sf_l 9556 Seepioph a jonesi symbiont

Proteobacteria Garnmaproteobacteria SUP05 Unclassified sf_l 8605 bacterioplankton clone ZA2525c

Proteobacteria Gammaproteobacteria SUP05 Unclassified sf_l 8654 inactive deep-sea hydro thermal vent chimneys clone IheB2-31

Proteobacteria Gammaproteobacteria SUP05 Unclassified sf_l 8965 B thymodiolus thermophilus gill symbiont Proteobacteria Gammaproteobacteria uranium waste clones Unclassified sf_l 8231 uranium waste soil clone JG30a-KF-21 Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8339 water 5 m downstream manure clone 35ds5 Proteobacteria Gammaproteobacteria Ellin307/WD2124 Unclassified sf_l 8532

Proteobacteria Garnmaproteobacteria Hlm307/WD2124 Unclassified sf_l 9458 uranium mining waste pile clone JG37-AG-94 proteobacterium

Proteobacteria Garnmaproteobacteria SAR86 Unclassified sf_l 8962 bacterioplankton clone AEGEAN _234 Proteobacteria Gammaproteobacteria Legionellales Unclassified sf_3 8587 Mars Odyssey Orbiter and encapsulation facility clone T5-3

Proteobacteria Gammaproteobacteria GAO cluster Unclassified sf_l 9468 activated sludge done SBRL2.40

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_4 8855

Proteobacteria Unclassified Unclassified Unclassified sf_8 9558

Proteobacteria Gammaproteobacteria Oceanospirillales Unclassified sf_3 8230

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8245

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8883

Proteobacteria Garnmaproteobacteria Unclassified Unclassified sf_3 9044 hydrothermal sediment done AF420370 Proteobacteria Gammaproteobacteria Thiotrichales Unclassified sf_l 8323 hydrothermal sediment done AF420363 Proteobacteria Alphaproteobacteria Unclassified Unclassified sf_6 8780 uranium mining mill tailing done GR-296.II.89

GR-296.n.89

Proteobacteria Gammaproteobacteria Oceanospirillales Unclassified sf_3 8327 Arctic sea ice ARK10148

Proteobacteria Garnmaproteobacteria Unclassified Unclassified sf_3 8606

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8714 Marinobacter hydrocarbonoclasticus str. ATCC

27132T

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 8959 bacterioplankton done AEGEAN_133 Proteobacteria Gammaproteobacteria Alteromonadales Unclassified sf_l 8483 Rheinheimera baltica str. OS140 Baltic # 166 Proteobacteria Gammaproteobacteria Shewanella Unclassified sf_l 9344 Shewanella algae str. ATCC 51192

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 9367 USA: Pacific Ocean seawater Naha Vents Hawaii isolate str. PV-4

Proteobacteria Gammaproteobacteria Unclassified Unclassified sf_3 9473 Arctic pack ice; northern Fram Strait; 80 31.1 N;

01 deg 59.7 min E done AR DMS-58

Proteobacteria Gammaproteobacteria Enterobacteriales Unclassified sf_l 8430 Salmonella bongori str. JEO 4162

Proteobacteria Deltaproteobacteria Desulfovibrionales Unclassified sf_l 9828 termite gut homogenate done Rs-M89

proteobacterium

Proteobacteria Deltaproteobacteria Myxococcales Unclassified sf_l 10092 heavy metal<ontainirtated soil done al3134 Proteobacteria Deltaproteobacteria Myxococcales Unclassified sf_l 10259

Proteobacteria Deltaproteobacteria Unclassified Unclassified sf_7 10048

Proteobacteria Deltaproteobacteria Unclassified Unclassified sf_9 10049 DCP-dechlorinating consortium done SHA-72 Proteobacteria Deltaproteobacteria Unclassified Unclassified sf_9 9760 deep marine sediment done MB-A2-137 Proteobacteria Deltaproteobacteria Unclassified Unclassified sf_9 9784 Antarctic sediment done LH5_30

Proteobacteria Deltaproteobacteria Unclassified Unclassified sf_9 9798 uranium mill tailings soil sample clone GuBH2-

AD/TzT-67 proteobacterium

Proteobacteria Deltaproteobacteria Unclassified Unclassified sf 9 9876 deep marine sediment clone MB-B2-106 Proteobacteria Deltaproteobacteria EB1021 group Unclassified sf 4 9884 forested wetland clone RCP2-62

Proteobacteria Deltaproteobacteria AMD clone group Unclassified sf 1 10084 acid mine drainage clone AS6

Proteobacteria Deltaproteobacteria Desulfuromonadales Unclassified sf 1 10076 Great Artesian Basin clone G13

Proteobacteria Deltaproteobacteria dechlorinating Unclassified sf_l 9959 forested wetland clone FW110

clone group

Proteobacteria Deltaproteobacteria EB1021 group Unclassified sf 4 10024 hydrothermal sediment clone AF420338 Proteobacteria Deltaproteobacteria AMD clone group Unclassified sf 1 9678 coal effluent wetland clone RCP185

Proteobacteria Deltaproteobacteria Desulfobacterales Unclassified sf 4 9951 forested wetland clone FW13

Proteobacteria Deltaproteobacteria Unclassified Unclassified sf 9 9738 marine methane seep clone 1513

Proteobacteria Deltaproteobacteria AMD clone group Unclassified sf 1 9945 acid mine drainage clone BA18

Proteobacteria Deltaproteobacteria Desulfobacterales Unclassified sf 3 9813 hydrothermal sediment clone AF420340 Proteobacteria Deltaproteobacteria Unclassified Unclassified sf_9 9890 termite gut homogenate clone Rs- 70

proteobacterium

Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sf 1 10543 hydrothermal vent clone PVB_10

Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sf_l 10427 hydrothermal vent 9 degrees North East Rise

Pacific Ocean clone

CH3_17_BAC_16SrRNA_9N_EPR

Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sf 1 10475 hydrothermal sediment clone AF420359 Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sf_l 10480 Paralvinelk palmijbrmis mucus secretions clone

P. palm C 84 proteobacterium

Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sU 10489 S17sBacl6 complete clone

Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sf_l 10497 UASB reactor granular sludge clone PD-UASB-2 proteobacterium

Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sU 10530 hydrothermal vent 9 degrees North East Rise

Pacific Ocean clone

CH5_6_BAC_16SrRNA_9N _^ EPR

Proteobacteria Unclassified Unclassified Unclassified sfJ20 2520

Proteobacteria Deltaproteobacteria Unclassified Unclassified sf_9 244 deep marine sediment clone MB-C2-152 Proteobacteria Deltaproteobacteria AMD clone group Unclassified sf 1 3084 coal effluent wetland clone RCP216

Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae sU 8999 Photobacterium leiognathi str. LN101

Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae sf 1 8665 Vibrio gallicus str. OP 107867; HT 3-3 Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae sf 1 8267 Vibrio pomeroyi str. LMG 20537

Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae sf 1 8798 Vibrio aestuari nus str. KT0901

Proteobacteria Gammaproteobacteria Vibrionales Vibrionaceae sf 1 8888 Vibrio aestuarianus str. 01/151

Proteobacteria Alphaproteobacteria Bradyrhizobiales Xanthobacteraceae sf 1 6660 Azorhizobium caulinodans str. ORS 571 Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 9167 pea aphid symbiont clone APe4_38

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 8689 Dyemonas todaii str. XD10

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf_3 9332 wetland ecosystem constructed to remediate mine drainage isolate str. WJ2 WJ2

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 8392 penguin droppings sediments clone D2-14 Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 8983 Iron oxidizing strain ES-1

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf_3 9031 municipal wastewater treatment bioreactor clone LB-P bacterium

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 9320 Waste-gas biofilter clone BIyi3

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 8577 Xanthinurias axonopodis pv. dtri str. MA Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 9569

(continued)

TABLE 3. (CONTINUED)

Phylum Class Order Family S-F* Taxon Hp Representative species

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 8538

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf_3 8563 Pseudoxanthomonas mexicana str. AMX 26B

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf_3 9270 Stenotrophomonas rhizopMa str. e-plO

Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae sf_3 9286 Stenotrophomonas maltophilia str. LMG 11104

SPAM Unclassified Unclassified Unclassified sf 1 705 uranium tailings soil clone Sh765B-AG-45

SPAM Unclassified Unclassified Unclassified sf 1 738 uranium mining waste clone JG34-KF-252

Spirochaetes Spirochaetes Spirochaetales Leptospiraceae sf_3 6496 Leptospira interrogans serovar Copenhagen! str.

Fiocruz Ll-130

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf_l 6459 Spirochaeta sp. str. BHI80-158

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf 3 6558 Spironema culicis str. BR91

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sU 6526 Treponema sp. str. 7CPL208

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf_l 6479 Treponema sp

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sU 6580 Treponema sp. str. HI:C:BA213

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sU 6458 termite gut clone kS34

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf 1 6494 termite gut homogenate clone Rs-C47 sp. Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf 1 6562 forested wetland clone RCPl-96

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf_l 6507 termite gut clone NkS-Ste2

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf 1 6476 termite gut clone NkS50

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf 1 6488 Treponema primitia str. ZAS-1

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf_l 6490 termite gut homogenate clone BCf4-14 Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf 1 6491 termite gut homogenate clone BCf8-03 Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf 1 6506 termite gut homogenate clone Rs-J58 sp. Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sU 6508 termite hindgut clone mpsp2

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sU 6523 termite gut homogenate clone Rs-J64 sp. Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf_l 6565 termite gut clone NkS-Oxy25

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae sf_l 6571 Mixotricha paradoxa is flagellate hindgut

Mastotermes darwiniensds clone mp4 of

Synergistes Unclassified Unclassified Unclassified sf 3 117 termite gut homogenate clone Rs-D89 Synergistes Unclassified Unclassified Unclassified sf_3 353 UASB reactor granular sludge clone PD-UASB-

13 G+C

Synergistes Unclassified Unclassified Unclassified sf_3 60 Flexistipes sp. str. E3_33

Synergistes Unclassified Unclassified Unclassified sf 3 601 terephthalate-degrading consortium clone TA19

Synergistes Unclassified Unclassified Unclassified sf_3 719 Synergistes sp. PI str. P4G_18

Synergistes Unclassified Unclassified Unclassified sf 3 740 swine intestine clone p-4292-4Wa3

Synergistes Unclassified Unclassified Unclassified sf 3 808 oral cavity clone BH017

Termite group 1 Unclassified Unclassified Unclassified sf_2 437 termite gut homogenate clone Rs-D43 group

Thermodesulfobacteria Thermodesulfobacteria Thermodesulfobacteriales Thermodesulfobacteriaceae sf 1 667 Geothermobacterium Jerrireducens

Thermotogae Thermotogae Thermotogales Thermotogaceae sf 4 . 51 Thermosipho sp. str. MV1063

TM6 Unclassified Unclassified Unclassified sf 1 9803 forest soil clone S1204

TM7 Unclassified Unclassified Unclassified sf 1 5177

TM7 TM7-3 Unclassified Unclassified sf_l 8155 oral periodontitis clone EW086

TM7 TM7-3 Unclassified Unclassified sf_l 2697 midgut homogenate Pachnoda ephippiata larva clone PeM47

TM7 Unclassified Unclassified Unclassified sf 1 3025

Unclassified Unclassified Unclassified Unclassified sf_93 925 4MB-degrading consortium clone UASB_TL26

Unclassified Unclassified Unclassified Unclassified sf 106 243 hot spring clone OFB25

Unclassified Unclassified Unclassified Unclassified sf 160 485 thermal spring mat clone OlaA90

Unclassified Unclassified Unclassified Unclassified sf 160 226

Unclassified Unclassified Unclassified Unclassified sf 160 333

Unclassified Unclassified Unclassified Unclassified sf 160 651

Unclassified Unclassified Unclassified Unclassified sf 160 6430

Unclassified Unclassified Unclassified Unclassified sf 160 6456

Unclassified Unclassified Unclassified Unclassified sf_160 6360

Unclassified Unclassified Unclassified Unclassified sf 140 6355

Unclassified Unclassified Unclassified Unclassified sf 160 7444

Unclassified Unclassified Unclassified Unclassified sf_160 7767

Unclassified Unclassified Unclassified Unclassified sf 160 10012

Unclassified Unclassified Unclassified Unclassified sf_95 2545 anaerobic sludge isolate str. JE

Unclassified Unclassified Unclassified Unclassified sf 160 2488

Unclassified Unclassified Unclassified Unclassified sf_156 4291 Mono Lake at depth 35m station 6 July 2000 clone ML635J-21 G+C

Unclassified Unclassified Unclassified Unclassified sf 160 4410

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Unclassified sf 4 169 anoxic marine sediment clone LD1-PA26

Verrucomicrobia Unclassified Unclassified Unclassified sf 3 40 Elbe river clone DEV055

Verrucomicrobia Unclassified Unclassified Unclassified sf 3 486 Elbe river clone DEV045

Verrucomicrobia Unclassified Unclassified Unclassified sf 5 686 hydrothermal vent sediment clone a2b018

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Unclassified sf 3 11 sludge clone H2

Verrucomicrobia Unclassified Unclassified Unclassified sf_4 288 Prosthecobacter dejongeii

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Unclassified sf 3 792 termite gut homogenate clone Rs-P07 bacterium -

VeiTucornicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobia SD 5 sf 1 530 anoxic marine sediment done LD1-PB20

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobia SD 5 sf 1 533 anoxic marine sediment done LD1-PB12

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobia SD 5 sf 1 547 anoxic marine sediment done LD1-PB1

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobia SD 5 sf 1 629 anoxic marine sediment done LD1-PA50

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobia SD 7 sU 446 anoxic marine sediment done LD1-PA34

Verrucomicrobia Vermcomicrobiae Verrucomicrobiales Verrucomicrobia SD 7 sf 1 559 anoxic marine sediment done LD1-PA20

Vemicomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobia SD 7 sf 1 760 Mono lake done ML316M-1

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobiaceae sf 7 29 FucopMus fucoidanolyticus str. SI-1234

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Verrucomicrobiaceae sf 6 871

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Xiphinematobacteraceae sf 3 888 Candida tus Xiphinematobacter brevicolli

WS3 Unclassified Unclassified Unclassified sf_3 95 marine sediment above hydrate ridge done

Hyd24-32

WS3 Unclassified Unclassified Unclassified sf 1 2537 anoxic marine sediment done LD1-PA39

WS5 Unclassified Unclassified Unclassified sL2 8119 hydrothermal vent sediment done a2b013

°S-F, Subfamily identification; 'Taxon ID, PhyloChip Taxon identification number; Representative species, Taxon bacterial species identifier.

TABLE 4. BACTERIAL TAXA WITH SIGNIFICANT DIFFERENCES IN RELATIVE ABUNDANCE BETWEEN COPD PATIENT GROUP 1 (<6 INTUBATION DAYS)

AND GROUP 2 (>16 INTUBATION DAYS)

Fluorescence difference (Group 1

Phylum Class Order Family s-r Taxon ID* Representative species' p-value q-value Group 2,

Finnicutes Symbiobacteria Symbiobacterales Unclassified 1 77 · thermal soil clone YNPFFP9 <0.001 <0.01 1264

Proteobacteria Deltaproteobacteria Unclassified Unclassified 9 244 deep m 1 arine sediment clone MB- <0.02 <0.05 1048

Chloroflexi Anaerolineae Unclassified Unclassified 9 375 forest soil clone C043 <0.02 <0.05 1873

Proteobacteria Deltaproteobacteria Desulfuromonadales Geobacteraceae 1 482 tricMoroethene-cmtaminated <0.01 <0.05 1092 site clone FTLM205

proteobacterium

OP10 CH21 cluster Unclassified Unclassified 1 514 sludge clone SBRA136 <0.01 <0.05 1081

Chlorobi Unclassified Unclassified Unclassified 8 636 benzene-degrading nitrate- <0.01 <0.05 1475 reducing consortium clone

Cart-N3 bacterium

Unclassified Unclassified Unclassified Unclassified 160 651 <0.01 <0.05 1750

Chloroflexi Unclassified Unclassified Unclassified 7 757 DCP-decrdorinating consortium <0.001 <0.01 1619 clone SHA-8

Natronoanaerobium Unclassified Unclassified Unclassified 1 769 fjord ikaite column clone un-c23 <0.001 <0.01 1305

Finnicutes Clostridia Qostridiales Peptococc/ 11 940 Veillonella dispar str. DSM 20735 <0.01 <0.05 1150

Addaminococc

OP9/JS1 OP9 Unclassified Unclassified 1 969 DCP-dechlorinating consortium <0.02 <0.05 1190 clone SHA-1

Finnicutes Bacilli Bacillales Bacillaceae 1 1050 Bacillus firmus CV93b <0.001 <0.05 1746

Actinobacteria Actinobacteria Unclassified Unclassified 1 1898 termite gut homogenate clone Rs- <0.01 <0.05 1906

]10 bacterium

AD3 Unclassified Unclassified Unclassified 1 2338 uranium mining waste pile soil <0.001 <0.01 1148 clone JG30-KF-C12

Chloroflexi Dehalococcoidetes Unclassified Unclassified 1 2339 uranium mill tailings soil sample <0.02 <0.05 1532 clone Sh765B-TzT-20

bacterium

Chloroflexi Unclassified Unclassified Unclassified 1 2534 forest soil clone S085 <0.001 <0.01 1193

Finnicutes Clostridia Qostridiales Lachnospiraceae 5 2668 termite gut homogenate clone Rs- <0.01 <0.05 2395

G40 bacterium

Finnicutes Qostridia Qostridiales Peptostreptococcaceae 5 2694 oral periodontitis clone FX028 <0.02 <0.05 1338

Finnicutes Clostridia Qostridiales Peptostreptococcaceae 5 2714 termite gut homogenate clone Rs- <0.01 <0.05 2061

N27 bacterium

Finnicutes Qostridia Qostridiales Peptostreptococcaceae 5 2729 DCP-decrdorinating consortium <0.01 <0.05 1402 clone SHA-58

Finnicutes Qostridia Qostridiales Peptostreptococcaceae 5 2797 Isolation and identification <0.01 <0.05 1611 hyper-ammonia producing

swine storage pits manure

Finnicutes Qostridia Qostridiales Peptostreptococcaceae 5 2805 oral periodontitis clone FX033 <0.02 <0.05 1625

Finnicutes Qostridia Qostridiales Lachnospiraceae 5 2834 Butyrwibrw fibrisolvens str. OB156 <0.01 <0.05 1005

Finnicutes Qostridia Qostridiales Lachnospiraceae 5 2994 termite gut clone Rs-L15 <0.001 <0.01 3929

Firmicutes Clostridia Qostridiales Clostridiaceae 12 3021 Clostridium caminithermale str. <0.01 <0.05 1944

DVird3

Firmicutes Clostridia Qostridiales Lachnospiraceae 5 3038 swine intestine clone p-1594-c5 <0.01 <0.05 1363 Firmicutes Clostridia Clostridiales Lachnospiraceae 5 3059 Butyrwibrio fibrisolvens str. NCDO <0.01 <0.05 1069

2249

Firmicutes Clostridia Qostridiales Lachnospiraceae 5 3060 termite gut homogenate clone Rs- <0.001 <0.01 3703

B14 bacterium

Firmicutes Clostridia Qostridiales Lachnospiraceae 5 3076 Clostridium nexile <0.001 <0.01 1395 Firmicutes Clostridia Qostridiales Clostridiaceae 12 3077 Clostridium glycolicum str. DSM <0.01 <0.05 1953

1288

Firmicutes Clostridia Qostridiales Lachnospiraceae 5 3171 Lachnospira pectinoschiza <0.01 <0.05 1398 Firmicutes Clostridia Qostridiales Peptostreptococcaceae 5 3182 termite gut homogenate clone Rs- <0.01 <0.05 1107

Q64 bacterium

Firmicutes Bacilli Lactobacillales Streptococcaceae 1 3250 Streptococcus bovis str. B315 <0.001 <0.01 4284 Firmicutes Bacilli Lactobacillales Streptococcaceae 3251 Streptococcus cristatus str. ATCC <0.01 <0.05 3986

¹ 51100

Firmicutes Bacilli Lactobacillales Streptococcaceae 3253 derived cheese sample clone <0.02 <0.05 2680

¹ 32CR

Firmicutes Bacilli Bacillales Staphylococcaceae 3258 Staphylococcus auricularis str. <0.01 <0.05 1525

¹ MAFF911484 ATCC33753T

Firmicutes Bacilli Lactobacillales Enterococcaceae 3261 Enterococcus mundtii str. LMG <0.02 <0.05 2560

¹ 10748

Firmicutes Bacilli Bacillales Bacillaceae 1 3283 Bacillus niacini str. IF015566 <0.01 <0.05 1201 Firmicutes Bacilli Bacillales Staphylococcaceae 1 3284 <0.01 <0.05 1347 Firmicutes Bacilli Bacillales Caryophanaceae 3285 Caryophanon latum str. DSM <0.01 <0.05 1499

O ¹ 14151

GO Firmicutes Bacilli Lactobacillales Streptococcaceae 3287 tongue dorsum scrapings clone <0.01 <0.05 3582

¹ FP015

Firmicutes Bacilli Lactobacillales Enterococcaceae 3288 Isolation and identification <0.01 <0.05 2528

¹ hyper-ammonia producing

swine storage pits manure

Firmicutes Bacilli Lactobacillales Streptococcaceae 1 3290 Streptococcus mitis str. Sm91 <0.01 <0.05 3971 Firmicutes Bacilli Bacillales Paenibacillaceae 3299 BrevibacUlus borstelensis str. LMG <0.02 <0.05 1035

¹ 15536

Firmicutes Bacilli Lactobacillales Streptococcaceae 3313 Streptococcus salivarius str. ATCC <0.001 <0.01 3189

¹ 7073

Firmicutes Bacilli Lactobacillales Enterococcaceae 3318 Enterococcus ratti str. ATCC <0.02 <0.05 2272

¹ 700914

Firmicutes Bacilli Lactobacillales Aerococcaceae 3323 Trichococcus flocculifirrmis str. <0.01 <0.05 1431

¹ DSM 2094

Firmicutes Bacilli Lactobacillales Aerococcaceae 1 3326 Nostocoida limicola I str. Ben206 <0.01 <0.05 2363 Firmicutes Bacilli Bacillales Bacillaceae 1 3328 Pseudobac lus carolinae <0.001 <0.01 2370 Firmicutes Bacilli Lactobacillales Lactobacillaceae 3330 Lactobacillus kitasatonis str. <0.02 <0.05 1389

¹ KM9212

Firmicutes Bacilli Bacillales Sporolactobacillaceae 1 3365 Bacillus sp. clone ML615J-19 <0.001 <0.01 1757 Firmicutes BariUi Lactobacillales Aerococcaceae 1 3386 feedlot manure clone B87 <0.01 <0.05 2321 Firmicutes Bacilli Lactobacillales Enterococcaceae 3392 Vagococcus lutrae str. mll34/97/ <0.001 <0.01 1976

¹ 1; CCUG 39187

(continued)

TABLE 4. (CONTINUED)

Fluorescence difference

(Group 1 -

Phylum Class Order Family S-P Faxo iD ^b Representative species' ¹ γ-value Rvalue Group 2)

Firmicutes Bacilli LactobadUales Strep tococcaceae 1 3397 Streptococcus macedonicus str. <0.01 <0.05 4011

ACA-DC 206 LAB617

Firmicutes Bacilli Lactobacillales Lactobatillaceae 1 3418 Lactobacillus subsp. aviarius <0.01 <0.05 3036

Firmicutes Bacilli Bacillales BadUaceae 1 3419 Bacillus algicola str. KMM 3737 <0.01 <0.05 1590

Firmicutes Bacilli Lactobacillales Streptococcaceae 1 3422 Streptococcus thermoph us str. <0.01 <0.05 3243

DSM 20617

Firmicutes Bacilli Lactobacillales Enterococcaceae 1 3433 Tetragenococcus muriaticus <0.01 <0.05 2715

Firmicutes Bacilli Lactobacillales Streptococcaceae 1 3446 Streptococcus bouts str. HJ50 <0.01 <0.05 3846

Firmicutes Bacilli Lactobacillales Unclassified 1 3481 <0.01 <0.05 2102

Firmicutes Bacilli Bacillales Bacillaceae 1 3489 Bacillus sOoestris str. SAF -010 <0.001 <0.01 1206

Firmicutes Bacilli Bacillales Bacillaceae 1 3492 Bacillus subtUis str. IAM 12118T <0.01 <0.05 1320

Firmicutes Bacilli Bacillales Staphylococcaceae 1 3494 Micrococcus luteus B-P 26 <0.01 <0.05 1334

Firmicutes Bacilli Lactobacillales Leuconostocaceae 1 3497 Weissella koreensis S-5673 <0.02 <0.05 1457

Firmicutes Bacilli Lactobacillales Streptococcaceae 1 3499 Streptococcus consteUatus str. <0.01 <0.05 4476

ATCC27823

Firmicutes Bacilli Lactobacillales Aerococcaceae 1 3504 Marinilactibacillus psychrotolerans <0.01 <0.05 1505 str. Q21

Firmicutes Bacilli Bacillales Bacillaceae 1 3517 Planococcus maritimus str. TF-9 <0.01 <0.05 1358

Firmicutes Bacilli Lactobacillales Lactobacillaceae 1 3521 Pediococcus inopinatus str. DSM <0.001 <0.05 1122

Firmicutes Bacilli Lactobacillales Lactobacillaceae 1 3526 Lactobacillus s kei <0.02 <0.05 1609

Firmicutes Bacilli Bacillales Staphylococcaceae 1 3545 <0.01 <0.05 1372

Firmicutes Bacilli LactobadUales lactobacillaceae 1 3547 Lactobacillus frumenti str. TMW <0.01 <0.05 1491

Firmicutes Bacilli Bacillales Bacillaceae 1 3550 Bacillus megaterium str. QM B1551 <0.001 <0.05 1620

Firmicutes Bacilli Lactobacillales Aerococcaceae 1 3553 Desemzia incerta str. DSM 20581 <0.001 <0.01 1553

Firmicutes Bacilli Lactobacillales Streptococcaceae 1 3560 Streptococcus gallinaceus str. <0.001 <0.01 2835

CCUG 42692

Firmicutes Bacilli Lactobacillales Lactobacillaceae 1 3566 Lactobacillus pontis str. LTH 2587 <0.01 <0.05 2320

Firmicutes Bacilli Bacillales Staphylococcaceae 1 3569 Staphylococcus s prophyticus <0.01 <0.05 1391

Firmicutes Bacilli Lactobacillales Streptococcaceae 1 3588 Streptococcus downei str. ATCC <0.01 <0.05 2440

Firmicutes Bacilli Bacillales Bacillaceae 1 3589 Bacillus senegalensis str. RS8; OP <0.02 <0.05 1198

106 669

Firmicutes Bacilli Bacillales Staphylococcaceae 1 3592 Staphylococcus caprae str. DSM <0.01 <0.05 1322

Firmicutes Bacilli Bacillales Staphylococcaceae 1 3605 <0.01 <0.05 1472

Firmicutes Bacilli Bacillales Bacillaceae 1 3612 Bacillus schlegelii str. ATCC <0.01 <0.05 1224

43741T

Firmicutes Bacilli Bacillales Staphylococcaceae 1 3628 Staphylococcus haemolyticus str. <0.01 <0.05 1572

CCM2737

Firmicutes Bacilli Lactobacillales Streptococcaceae 1 3629 Streptococcus mutans str. UA96 <0.01 <0.05 1466

Firmicutes Bacilli BaciUales Halobadllaceae 3633 Bacillus clausii str. GMBAE 42 <0.001 <0.01 2363

Firmicutes Bacilli Lactobacillales Lactobacillaceae 3634 Lactobacillus letivazi str. JCL3994 <0.01 <0.05 1586

Firmicutes Bacilli BaciUales Staphylococcaceae 3638 Staphylococcus sp str. AG-30 <0.01 <0.05 1359

Firmicutes Bacilli Bacillales Paenibacillaceae 3641 Bre bacUlus sp. MN 47.2a <0.02 <0.05 1735

Firmicutes Bacilli Bacillales Staphylococcaceae 3654 Staphylococcus pettenkorferi str. <0.01 <0.05 1310

B3117

Firmicutes Bacilli Bacillales Bacillaceae 3661 Bacillus sp. str. 221625.2 <0.01 <0.05 1593

Firmicutes Bacilli Bacillales Bacillaceae 3675 Bacillus mojavensis str. M-l <0.01 <0.05 1535

Firmicutes Bacilli Bacillales Staphylococcaceae 3684 Staphylococcus sciuri <0.02 <0.05 1324

Firmicutes Bacilli Bacillales Halobadllaceae 3702 Amphtbacillus xylanus str. DSM <0.01 <0.05 1523

6626

Firmicutes Bacilli Lactobacillales Lactobacillaceae 3703 Lactobacillus salivarius str. RA2115 <0.01 <0.05 1636

Firmicutes Bacilli Bacillales Bacillaceae 3706 Bacillus sonorensis str. RRL B- <0.02 <0.05 1324

23155

Firmicutes Bacilli Lactobacillales Strep tococcaceae 3722 Lactococcus 01403 subsp. lactis <0.001 <0.05 2673 str. IL1403

Firmicutes Bacilli Bacillales SporolactobadUaceae 3747 Bacillus sp. str. C-59-2 <0.001 <0.01 1959

Firmicutes Bacilli Lactobacillales Streptococcaceae 3753 Streptococcus suis str. 8074 <0.02 <0.05 3463

Firmicutes Bacilli Lactobacillales Lactobacillaceae 3767 Lactobacillus suebicus str. CECT <0.01 <0.05 2031

5917T

Firmicutes Bacilli Lactobacillales Lactobacillaceae 3768 Lactobacillus perolens str. L532 <0.001 <0.001 1593

Firmicutes Bacilli Bacillales Staphylococcaceae 3794 <0.01 <0.05 1324

Firmicutes Bacilli Bacillales Staphylococcaceae 3822 Staphylococcus succinus str. SB72 <0.01 <0.05 1358

Firmicutes Bacilli Bacillales Bacillaceae 3827 Bacillus acidogenesis str. 105-2 <0.01 <0.05 1996 o Firmicutes Bacilli Bacillales Bacillaceae 3831 Bacillus lichenifitrmis str. L-068 <0.01 <0.05 2057

Firmicutes Bacilli Lactobacillales Aerococcaceae 3833 Carnobacterium alterfunditum <0.001 <0.01 2781

Firmicutes Bacilli Lactobacillales Aerococcaceae 3840 Trichococcus pasteurii str. KoTa2 <0.001 <0.01 2656

Firmicutes Bacilli Lactobacillales Streptococcaceae 3869 Streptococcus equi subsp. <0.01 <0.05 1766 zooepidemicus str. Tokyol291

subsp.

Firmicutes Bacilli Bacillales Bacillaceae 3900 Bacillus licheniformis str. DSM 13 <0.01 <0.05 1261

Firmicutes Bacilli Lactobacillales Streptococcaceae 3906 Streptococcus bovis str.ATCC · <0.001 <0.01 4284

43143

Firmicutes Bacilli Bacillales Bacillaceae 3909 Bacillus subtilis subsp. Marburg <0.01 <0.05 1367 str. 168

Firmicutes Bacilli Bacillales Bacillaceae 3918 Bacillus subtilis <0.001 <0.01 1486

Firmicutes Mollicutes Anaeroplasma tales Erysipelotrichaceae 3 3965 TCE<cmtaminated site done <0.01 <0.05 1844 ccslm238

Firmicutes Mollicutes Anaeroplasma tales Erysipelotrichaceae 3 3981 phototrophic sludge done PSB- <0.01 <0.05 1361

M-3

Firmicutes Clostridia Clostridiales Qostridiaceae 12 4180 termite gut homogenate done Rs- <0.01 <0.05 1383

M23 bacterium

Firmicutes Clostridia Unclassified Unclassified 7 4216 <0.001 <0.01 2447

Firmicutes Clostridia Clostridiales Qostridiaceae 12 4266 termite gut homogenate done Rs- <0.01 <0.05 1302

M86 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae 5 4281- granular sludge done <0.001 <0.05 1333

UASB_brew_B86

Firmicutes gut clone group Unclassified Unclassified 1 4298 human mouth clone P4PA_66 <0.01 <0.05 1991

TABLE 4. (CONTINUED)

Fluorescence different (Group 1

Phylum Class Order Family S-f* Taxon iD ^b Representative species' ¹ p-value q-value Group 1

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4306 UASB reactor granular sludge <0.01 <0.05 1483 clone PD-UASB-4 bacterium

Finnicutes Clostridia Clostridiales Clostridiaceae 12 4321 termite gut homogenate clone Rs- <0.01 <0.05 1362

C76 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae 5 4331 granular sludge clone <0.01 <0.05 1091

UASB_brew_B84

Fi micutes Clostridia Clostridiales Clostridiaceae 12 4339 Clostridium chauvoei str. ATCC <0.02 <0.05 1542

10092T

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4369 termite gut homogenate clone Rs- <0.01 <0.05 1579

N73 bacterium

Natronoanaerobium Unclassified Unclassified Unclassified 1 4377 Mono Lake at depth 35 m station <0.01 <0.05 1585

6 July 2000 clone ML635J-65

G + C

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4418 termite gut homogenate clone Rs- <0.02 <0.05 1173

H18 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae 5 4434 termite gut homogenate clone Rs- <0.01 <0.05 1298

Kll bacterium

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4475 termite gut homogenate clone Rs- <0.001 <0.01 1853

N02 bacterium

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4477 termite gut homogenate clone Rs- 0.01 <0.05 1505

N85 bacterium

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4507 termite gut homogenate clone Rs- <0.01 <0.05 1169

N21 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae 5 4510 termite gut homogenate clone Rs- <0.02 <0.05 1896

Q53 bacterium

Firmicutes Clostridia Clostridiales Lachnospiraceae 5 4512 granular sludge clone <0.01 <0.05 1006

UASB_brew_B25

Finnicutes Clostridia Clostridiales Lachnospiraceae 5 4514 termite gut homogenate clone Rs- <0.001 <0.05 1800

B34 bacterium

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4524 termite gut clone Rs-093 <0.02 <0.05 1269

Firmicutes Clostridia Clostridiales Lachnospiraceae 5 4533 termite gut homogenate clone Rs- <0.01 <0.05 1671

N06 bacterium

Firmicutes Unclassified Unclassified Unclassified 8 4536 Mono Lake at depth 35 m station <0.01 <0.05 1005

6 July 2000 clone ML635J-14

G + C

Firmicutes Clostridia Clostridiales Lachnospiraceae 5 4540 termite gut homogenate clone Rs- <0.01 <0.05 1962

M18 bacterium

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4598 Clostridium sardiniense six. DSM <0.02 <0.05 1253 ouu

Firmicutes Clostridia Clostridiales Clostridiaceae 12 4607 Clostridium novyi str. NCTC538 <0.01 <0.05 1082

Firmicutes Clostridia Clostridiales Lachnospiraceae 5 4613 rumen clone 3C0d-3 <0.02 <0.05 1321

Firmicutes gut clone group Unclassified Unclassified 1 4616 rumen clone F23-C12 <0.01 <0.05 2628

Firmicutes Clostridia Clostridiales Qostridiaceae 12 4622 termite gut clone Rs-L36 <0.01 <0.05 1112

Finnicutes Clostridia Qostridiales Qostridiaceae 12 4638 <0.01 <0.05 2515

Cyanobacteria Unclassified Unclassified Unclassified 9 5038 Rumen isolate str. YS2 <0.001 <0.01 1724

Bacteroidetes Bacteroidetes Unclassified Unclassified 15 5481 marine sediment above hydrate <0.02 <0.05 2056 ridge clone Hyd89-72

bacterium

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae 19 5542 Cytophaga sp. 1-1787 <0.01 <0.05 2304

Bacteroidetes Bacteroidetes Bacteroidales Unclassified 15 5783 Mono Lake at depth 35 m station <0.01 <0.05 1032

6 July 2000 clone ML635J-15

bacterium

Bacteroidetes Bacteroidetes Bacteroidales Unclassified 15 5874 Paralvinella palmiformis mucus <0.02 <0.05 1805 secretions clone P. palm 53

bacterium

Bacteroidetes Sphingobacteria Sphingobacteriales Flexibacteraceae 19 6124 Flexibacter flexilis subsp. <0.001 <0.01 1387 pelliculosus str. IFO 16028

subsp.

Spirochaetes Spirochaetes Spirochaetales Spirochaetaceae 1 6459 Spirochaeta sp. str. BHI80-158 <0.001 <0.01 2558

Proteobacteria Deltaproteobacteria Desulfobacterales Unclassified 3 9813 hydrothermal sediment clone <0.01 <0.05 1453

AF420340

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae 5 9875 hydrothermal sediment clone <0.01 <0.05 1147

AF420354

Froteobacteria Deltaproteobacteria Desulfuromonadales Geobacteraceae 1 10171 <0.01 <0.05 1375

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfoarculaceae 2 10227 marine sediment clone Bolll <0.01 <0.05 1549

Proteobacteria Deltaproteobacteria Desulfobacterales Desulfobacteraceae 5 10319 sulf ate-reducdng habitat clone <0.01 <0.05 1235

SLM-CP-116

"S-F, Subfamily identification; ^b Taxan ID, FhyloChip Taxon identification number; ""Representative species, Taxon bacterial spedes identifier.

TABLE 5. CORE COMMUNITY OF BACTERIAL TAXA DETECTED IN ALL COPD PATIENTS DURING TREATMBNT FOR SEVERE EXACERBATIONS (REPRESENTATIVE SPECIBS

WITH A PROVEN ROLE IN MAMMALIAN PATHOGENESIS ARE HIGHUGHTED)

Phylum Class Order Family S-F* Taxon JD ^b Representative species'

Actinobacteria Actinobacteria Addimicrobiales Addimicrobiaceae sf 1 1749 forest soil clone DUNssu275 (-3A) (OTU#188)

Addobacteria Addobacteria Addobacteriales Addobacteriaceae sf 6 6362 grassland soil done DA052

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf 1 8578 Marinobacter lipolyticus str. SM-19

Proteobacteria Ganunaproteobacteria Alteromonadales Alteromonadaceae sf 1 9239 Arctic sea ice ARK10228

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf 1 8222

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf 1 8753 Idiomarina loihiensis str. GSP37

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf 1 9324 Pseudoalteromonas ruthenica str. M300

Proteobacteria Gammaproteobacteria Alteromonadales Alteromonadaceae sf 1 8579 Psychromonas profunda str. 2825

Proteobacteria Alphaproteobacteria Rickettsiales Anaplasmataceae sf 3 6648 Wolbachia sp

Proteobacteria Betaproteobacteria Burkholderiales Burkholderiaceae sf 1 7747

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae sf_3 10538 Arcobacter cryaeropMus

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae sf 3 10447 SulfurospirUlum deleyianum str. Spirillum 5175

Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae sf 3 10456 Campylobacter showae

Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae sf 1 6909 Brevundimonas diminuta str. DSM 1635

Proteobacteria Alphaproteobacteria Caulobacterales Caulobacteraceae sf 1 7436 Brevundimonas sp. str. FWC40

Cyanobacteria Cyanobacteria ChloToplasts Chloroplasts sf_5 5147 Emiliania huxleyi str. Plymouth Marine Laborator PML 92

Proteobacteria Gammaproteobacteria Legionellales Coxiellaceae sf 3 9198 uranium mining waste pile done KF-JG30-B15 KF-JG30-B1

Bacteroidetes Sphingobacteria Sphingobacteriales Crenotrichaceae sf 11 6267 Cilia-respiratory isolate str. 243-54

Proteobacteria Deltaproteobacteria Desulfovibrionales Desulfomicrobiaceae sf 1 10079 Oesulfomtcrobium baculatum str. DSM 1742

Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae sf 1 8504 Dysmicoccus neobrevipes symbiont

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10385

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10442 Helicobacter cetorum str. ΜΓΤ 99-5656

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10444 Helicobacter suncus str. Kaz-2

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10448 Helicobacter felis str. Dog-1

Proteobacteria Epsilonproteobacteria Campylobacterales Helicobacteraceae sf 3 10451 Helicobacter he mannii str. C4S

Spirochaetes Spirochaetes Spirochaetales Leptospiraceae sf 3 6496 Leptospira interrogans serovar Copenhageni str. Fiocruz Ll-

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf 3 8366 Psychrobacter frigidicok str. DSM 12411

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf 3 8838 Psychrobacter psychrophilus CMS 28

Proteobacteria Gammaproteobacteria Pseudomonadales Moraxellaceae sf 3 8727 Alkanindiges hongkongensis str. H U9

Proteobacteria Betaproteobacteria Nitrosomonadales Nitrosomonadaceae sf 1 7789

Finnicutes Clostridia Qostridiales Peptococc/Addaminococc sf_ll 992 anoxic bulk soil flooded rice microcosm done BSV43 done

Planctomycetes Planctomycetada Planctomycetales Pirellulae sf 3 4670

Proteobacteria Gammaproteobacteria Thiotrichales Pisdrickettsiaceae sf 3 9291 Methylophaga alcalica str. M39

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8691 Pseudomonas aeruginosa str. PAOl

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9056 Pseudomonas aeruginosa str. #47

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9068 Pseudomonas stutzeri str. A1501

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae . sf_l 9295

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9613 Pseudomonas flavescens str. B62

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9049 uranium mining mill tailing done GR-Sh2-34 GR-Sh2-34

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9469 d. Pseudomonas sp. done Llangefni 52

Proteobacteria Ganunaproteobacteria Pseudomonadales Pseudomonadaceae sf_l 9240 Pseudomonas fluorescens str. CHA0

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 9366 Arctic seawater isolate str. R7366

Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae sf 1 8755 Pseudomonas sp. SK-1-3-1

Bacteroidetes Sphingobacteria Sphingobacteriales Sphingobacteriaceae sf 1 5913 Sphingobacteriaceae str. FJlinl60

Proteobacteria Alphaproteobacteria Sphingomonadales Sphingomonadaceae sf 1 6663 Sphingopyxis flavimaris str. SW-151

Fiimicutes Bacilli Bacillales Thermoactinomycetaceae sf_l 3301 Thermoactinomyces sp. str. 700375

Thennodesulfobacteria Thennodesulfobacteria Thermodesulfobacteriales Thermodesulfobacteriaceae sf_l 667 Fjonesi

Proteobacteria Gam naproteobacteria Thiotrichales Thiotrichaceae sf 3 8752 Beggiatoa sp. str. MS-81-lc

Chloroflexi Unclassified Unclassified Unclassified s 818

Verrucomicrobia Unclassified Unclassified Unclassified sf 4 288 Prosthecobacter dejongeii

Synergistes Unclassified Unclassified Unclassified sf 3 117 termite gut homogenate clone Rs-D89

Synergistes Unclassified Unclassified Unclassified sf 3 719 Synergistes sp. PI str. P4GJL8

OP3 Unclassified Unclassified Unclassified sf 4 628 Q^contaminated groundwater clone GOUTB15

Unclassified Unclassified Unclassified Unclassified sf 160 485 thermal spring mat clone OlaA90

Unclassified Unclassified Unclassified Unclassified sf 160 226

Bacteroidetes KSA1 Unclassified Unclassified sf 1 5951 CFB group clone ML615J-4

Chloroflexi Anaerolineae Unclassified Unclassified sf 9 727 forest soil done S0208

Cyanobacteria Unclassified Unclassified Unclassified sf_8 5206

marine group A mgA-2 Unclassified Unclassified sf 1 6344 bacterioplankton clone ZA3648c

Unclassified Unclassified Unclassified Unclassified sf 160 6430

Proteobacteria Alphaproteobacteria Unclassified Unclassified sf 6 7575

TM7 TM7-3 Unclassified Unclassified sf 1 8155 oral periodontitis clone EW086

Proteobacteria Gamrnaproteobacteria uranium waste clones Unclassified sf 1 8747 uranium waste soil clone JG30-KF-CM35

Proteobacteria Gamrnaproteobacteria Unclassified Unclassified sf 3 9568 forested wetland clone RCP2-96

Proteobacteria Gamrnaproteobacteria SUP05 Unclassified sf 1 8605 bacterioplankton clone ZA2525c

Proteobacteria Gamrnaproteobacteria Unclassified Unclassified sf 3 8339 water 5 m downstream manure clone 35ds5

Proteobacteria Gamrnaproteobacteria Unclassified Unclassified sf 4 8855

Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sf_l 10480 Paralvinella palmiftrrmis mucus secretions cloneP. palm C 84 pro eobacterivan

Proteobacteria Epsilonproteobacteria Campylobacterales Unclassified sf_l 10530 hydrothermal vent 9 degrees North East

Rise PacificOcean done

CH5_6_BAC_16SrRNA_9N_EPR

Actinobacteria Actinobacteria Actinomycetales Unclassified sf 3 1687 Jonesia qtiinghaiensis str. DSM 15701

Actinobacteria Actinobacteria Actinomycetales Unclassified sf 3 1405 Arthrobacter ureafaciens str. DSM 20126

Firmicutes Clostridia Unclassified Unclassified sf 3 2373

Firmicutes Catabacter Unclassified Unclassified sf 1 4293 termite gut homogenate done Rs-QOl bacterium

Proteobacteria Gamrnaproteobacteria Xanthomonadales Xanthomonadaceae sf 3 8689 Dyemonas todaii str. XD10

Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Xiphinematobacteraceae sf 3 888 Candidatus Xtphinematobader breoicoUi

°S-F, Subfamily identification; 'Taxon ID, PhyloChip Taxon identification number; ^Representative species, Taxon bacterial species identifier.