FRAGMENTATION AND TAGMENTATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority under 35 U.S. C. § 119(e) to U.S. Provisional Patent Application No. 61/963,665, entitled "Ultra-Low Input System with Two Different End Recognition Sequences for DNA Fragmentation and Tagmentation" filed December 11, 2013, which is incorporated herein by reference in its entirety.

FIELD

[0002] The disclosed embodiments relate to compositions, methods and kits for generating DNA libraries from target DNA using transpososome complexes. At least some of the DNA fragments of such libraries are tagged at both ends, wherein the tag sequence at the 5 ' end of a fragment has a different nucleotide sequence than the tag present in the 3 ' end of that fragment. The disclosed DNA libraries are suitable for next generation sequencing application or other methods that rely on the use of tagged DNA fragments derived from the target genetic material.

BACKGROUND

[0003] Traditionally, Sanger dideoxy sequencing has been the preferred DNA sequencing method, although it typically requires extensive cloning or amplification of the target DNA. Preparing cloned or amplified DNA template is usually laborious, time-consuming, and expensive. Recently, next generation sequencing platforms have become available

commercially, for example, the SOLEXA™ (Illumina), 454 FLX™ or 454 TITANIUM™ (Roche) and the SOLID™ or Ion Torrent DNA Sequencer (Life Technologies/ Applied

Biosystems) systems.

[0004] Although the chemistry by which sequence information is generated may vary among different next generation sequencing platforms, all of the systems generate sequence data by simultaneously sequencing very large numbers of sequencing templates. So, rather than requiring genomic libraries of DNA clones in E. coli for Sanger sequencing, for example, it is now desirable to have in vitro systems for generating DNA fragment libraries comprising a collection or population of DNA fragments generated from target DNA in a sample, wherein the combination of all of the DNA fragments in the library exhibits sequences that are qualitatively and/or quantitatively representative of the sequence of the target DNA from which the DNA fragments were generated. In some cases, it is necessary to generate DNA fragment libraries comprising multiple genomic DNA fragment libraries, each of which is labeled with a different address tag, index or bar code to permit identification of the source of each fragment.

[0005] In general, these next generation sequencing methods require fragmentation of genomic DNA (gDNA) or double-stranded cDNA (prepared from RNA) into smaller single- stranded DNA (ssDNA) fragments and addition of tags to at least one strand or preferably both fragment strands.

[0006] Fragmentation of gDNA is one of the main steps in preparing samples for high- throughput sequencing. In general, current methods for generating DNA fragment libraries for next generation sequencing comprise fragmenting the target DNA that one desires to sequence (e.g. target DNA comprising genomic DNA or double-stranded cDNA after reverse transcription of RNA) using a sonicator, nebulizer, or a nuclease (for example, DNase I), and joining (e.g., by ligation) oligonucleotides consisting of adapters or tags to the 5' and 3' ends of the fragments. These traditional methods are laborious, time-consuming, and often result in DNA fragmentation that is either insufficient or too extensive. In either case, the yield of DNA fragments of useful size is low.

[0007] This difficulty has been overcome by controlled DNA fragmentation using oligonucleotide-transposase complexes, sometimes referred to as "transpososomes". Such complexes comprise a dimer of modified transposase Tn5 and a pair of Tn-5 binding double- stranded DNA (dsDNA) oligonucleotides containing a 19 base pair (bp) transposase-binding sequence (mosaic end, ME), or inverted repeat sequence (IR). Unlike DNase, a single molecule of which can generate numerous breaks in a target DNA, the transposase complex or

"transpososome" creates only one DNA cleavage per complex. Therefore, unlike with DNase I, the degree of DNA fragmentation is more easily controlled during transposase fragmentation.

SUMMARY

[0008] The present teachings are directed to compositions, methods and kits for making creating DNA libraries of tagged fragments, typically obtained from gDNA or double stranded cDNA.

[0009] According to certain embodiments, compositions for generating fragmented, tagged DNA libraries comprise at least one first oligonucleotide comprising at least one double- stranded portion, wherein the double-stranded portion comprises at least one first recognition end sequence; at least one second oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one second recognition end sequence; and a transposase. Certain compositions of the current teachings further comprise at least one third oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one third recognition end sequence; at least one fourth oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one fourth recognition end sequence;

[0010] According to certain embodiments, methods for generating a library of tagged DNA fragments comprise combining (a) a first oligonucleotide comprising at least one double- stranded portion, wherein the double-stranded portion comprises at least one first recognition end sequence; b) a second oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one second recognition end sequence; (c) at least one transposase; and (d) at least one target DNA sequence; and incubating the combination under conditions suitable for generating a DNA library wherein each DNA fragment comprises at least two different recognition end sequences, at least two sequences complimentary to two different recognition end sequences, or at least one recognition end sequence and at least one sequence that is complimentary to a different recognition end sequence. In certain embodiments, the target DNA is gDNA or double-stranded cDNA.

[0011] Some method embodiments further comprise combining (a) a third

oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one third recognition end sequence; b) a fourth oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one fourth recognition end sequence; (c) at least one transposase. According to certain embodiments, the first oligonucleotide, the second oligonucleotide and the at least one transposase are incubated under conditions suitable a first transposome to form prior to adding the at least one target DNA sequence. In certain embodiments, the third oligonucleotide, the fourth oligonucleotide, and the at least one transposase are incubated under conditions suitable for a second transposome to form prior to adding the target DNA. [0012] In certain embodiments, (a) the at least one first recognition end sequence comprises SEQ ID NO: l, SEQ ID NO:3, or both; (b) the at least one second recognition end sequence comprises SEQ ID NO:2, SEQ ID NO:4, or both; or (c) (i) the at least one first recognition end sequence comprises SEQ ID NO: l, SEQ ID NO:3, or both, and (ii) the at least one second recognition end sequence comprises SEQ ID NO:2, SEQ ID NO:4, or both. In certain embodiments, the at least one first recognition end sequence comprises SEQ ID NO: l; the at least one second recognition end sequence comprises SEQ ID NO:2; the at least one third recognition end sequence comprises SEQ ID NO:3; and the at least one fourth recognition end sequence comprises SEQ ID NO:4.

[0013] Certain method embodiments further comprise amplifying at least one tagged DNA fragment using primers and the polymerase chain reaction (PCR). In certain embodiments, the PCR amplification comprises using at least one first primer and at least one second primer, wherein the 3' end portion of the first primer is complementary to the 3' end of the at least one first recognition end sequence of at least one DNA fragment and wherein the 3' end portion of the second primer comprises the sequence of at least a portion of the 5' end of the at least one first recognition end sequence of the at least one DNA fragment.

[0014] In certain embodiments, both the first primer and the second primer comprise 3' sequence that are the same as or are complimentary to at least a portion of the first recognition end sequence, the second recognition end sequence or both recognition end sequences. In certain embodiments, the first primer comprises SEQ ID NO:7 and the second primer comprises SEQ ID NO:8. In certain embodiments, the first primer, the second primer or both, further comprise sequencing tags comprising at least one of: Roche 454 sequencing tags, Illumina™ SOLEXA™ sequencing tags, Applied Biosystems' SOLID™ or Ion Torrent sequencing tags, the Pacific Biosciences' SMRT™ sequencing tags, Pollonator Polony sequencing tags, or Complete

Genomics sequencing tags.

[0015] Kits for generating tagged libraries of fragmented DNA are also disclosed.

According to certain embodiments, kits comprise: at least one first oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one first recognition end sequence; and at least one second oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one second recognition end sequence. Certain kit embodiments further comprise at least one third oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one third recognition end sequence; and at least one fourth

oligonucleotide comprising at least one double-stranded portion, wherein the double-stranded portion comprises at least one fourth recognition end sequence. In some kit embodiments, (a) the first recognition end sequence comprises SEQ ID NO: l, SEQ ID NO:3, or both, and (b) the second recognition end sequences comprises SEQ ID NO:2, SEQ ID NO:4, or both. In some kit embodiments, (a) the first recognition end sequence comprises SEQ ID NO: l; the second recognition end sequences comprises SEQ ID NO:2; the third recognition end sequence comprises SED ID NO:3; and the fourth recognition end sequence comprises SEQ ID NO:4.

[0016] Certain kits of the current teachings further comprise at least one adaptor and at least one oligonucleotide primer. In some embodiments, (a) the at least one adaptor comprises SEQ ID NO:5, SEQ ID NO:6, or both, (b) the at least one oligonucleotide primer comprises SEQ ID NO;7, SEQ ID NO:8, or both, or (c) (i) the at least one adaptor comprises SEQ ID NO:5, SEQ ID NO:6, or both, (ii) the at least one oligonucleotide primer comprises SEQ ID NO;7, SEQ ID NO:8, or both.

[0017] These and other features of the present teachings are set forth herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. These figures are not intended to limit the scope of the present teachings in any way.

[0019] FIG. 1 graphically depicts a disclosed method for generating DNA fragments form target DNA using a transposase (Tn5) and two different recognition end sequences (El and E2), sometimes referred to as transposon end sequences. First, a transposome complex comprising the transposase and the two different recognition end sequences is formed. Next, the transposome is combined with the target DNA and an in vitro transposition reaction occurs, resulting in DNA fragments with different recognition end sequences, El and E2, on each end (sometimes referred to as di-tagged dsDNA).

[0020] FIG. 2 graphically depicts a method for generating a DNA library using the exemplary tagged DNA fragments form FIG 1. First and second amplification primers, each comprising a different bar code sequence that the other primer, are annealed to the adaptor linked di-tagged DNA fragments. The fragments are then PCR amplified using DNA polymerase to generate a DNA library comprising bar coded (tagged) fragments.

[0021] FIG. 3 A shows an agarose gel of electrophoresed lambda DNA that was fragmented and tagged using an engineered transposase and two different recognition end sequences corresponding to that transposase according to a disclosed method. The fragments were amplified using one primer pair (Lane #1). Lane 2 shows the results obtained with a NEXTERA™ kit (Illumina) using a single ME and 4 amplification primers. .

[0022] FIG. 3B is a fragment size distribution printout of the tagged, fragmented DNA library shown in FIG. 3 A lane 1 obtained using an Agilent Bioanalyzer DNA 1000 kit.

[0023] FIG. 4 is an agarose gel of tagged DNA libraries generated according to Example 1. The first and last lanes shown molecular weight markers. Lane two (9948) is from a DNA library prepared from human gDNA. Lane 3 is a DNA library prepared from bacteriophage Lambda gDNA. Lane 4 is a DNA library prepared from Helicobacter pylori gDNA, and Lane 5 is a DNA library prepared from E. coli gDNA.

DETAILED DESCRIPTION

[0024] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings.

[0025] The instant application provides compositions, methods and kits for generating compositions related to tissue engineered constructs, including but not limited to, constructs designed to add volume at and/or below an incision or insertion site when certain constructs of the present teachings is implanted. For example, constructs designed according to certain disclosed tissue engineering methods that mimic the appearance or a nipple or a NAC when implanted. Also disclosed are methods for fabricating multilayered constructs, for fabricating constructs in a hydrogel scaffolds, and methods comprising bioprinting one or more cell type in or on a construct layer, or a hydrogel or other scaffold.

[0026] The term "adaptor" refers to a non-target nucleic acid sequence, generally DNA, that provides a means of addressing a nucleic acid fragment to which it is joined. In certain embodiments, an adaptor comprises a nucleotide sequence that permits identification, recognition, and/or molecular or biochemical manipulation of the DNA to which the adaptor is attached, for example, by providing a site for annealing an oligonucleotide, such as a primer for extension by a DNA polymerase, or an oligonucleotide for binding to a bead or other support, or for a ligation reaction. According to certain disclosed embodiments, one or more adaptors are incorporated in components of the fragmented DNA libraries of the current teachings. In certain embodiments, adaptors are used for polymerase-mediated amplification.

[0027] An "oligo" or "oligonucleotide" is a short piece of DNA or RNA that is typically single-stranded, but for purposes of the current teachings, an oligo may be double stranded or may be partially single stranded and partially double stranded.

[0028] The term "recognition end sequence" refers to the nucleotide sequence within the oligonucleotides of the current teachings (e.g., the first oligonucleotide, the second

oligonucleotide, and so forth) to which a transposase specifically binds to during transposome formation. Exemplary recognition end sequences of the current teachings comprise SEQ ID NOs: l-4.

[0029] A "transposase" is an enzyme that is involved in the transposition of DNA segments in the genome, for example, the movement of transposable elements during DNA mutagenesis. The term transposase as used herein may, in certain embodiments, further comprise related enzymes known as integrases. Repetitive transposable elements have been identified in the mammalian genome, among other places, and some repetitive elements are able to move within the genome (transposons and retrotransposons). Typically, DNA transposons move from one genomic location to another by a mechanism that is sometimes referred to as a "cut-and-paste" mechanism. Exemplary transposases include the DDE transposases (for example, the transposases of the maize Ac transposon, the Drosophila P element, bacteriophage Mu, Tn5 and TnlO, Mariner, IS 10, and IS50), and the synthetic Sleeping Beauty (SB) transposase.

[0030] In this application, the use of the singular includes the plural unless specifically stated otherwise. The use of "comprise", "contain", and "include", or modifications of those root words, for example but not limited to, "comprises", "contained", and "including", are not intended to be limiting. The term "and/or" means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, "X, Y and/or Z" can mean "X" or "Y" or "Z" or "X and Y" or "X and Z" or "Y and Z", of "X and Y and Z.". [0031] The section headings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. All literature and similar materials cited in this application, including patents, patent applications, articles, books, and treatises are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term's definition in this application, this application controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

Exemplary Embodiments

[0032] The following examples provide compositions, methods and kits for generating DNA libraries comprising fragments comprising different recognition end sequences on their respective ends, and in some embodiments, adaptor tags on one or both ends.

[0033] The current teachings provide new methods for preparing DNA libraries from gDNA or double-stranded cDNA targets. The DNA fragments in such libraries comprise two different recognition end sequences per DNA fragment created. Certain embodiments further comprise using two independently tagged primers for amplification of the DNA fragments. According to certain disclosed methods, random DNA fragments are generated each comprising two different recognition end sequences without regard to the sequences of the target DNA.

[0034] In one exemplary embodiment, depicted in FIG. 1, an aqueous composition comprising a transposase (shown as Tn5 in this example) and two different recognition end sequences (shown as El and E2) are incubated under conditions appropriate to form a transpososome, sometimes referred to as a transposome complex. The transpososome is combined with target DNA and under conditions suitable for in vitro transposition, the transpososomes bind the target DNA, transposition occurs (FIG. 1, middle panel), and DNA fragments comprising different recognition sequences on their ends are generated (FIG. 1, lower panel).

[0035] In certain embodiments, the oligonucleotides comprising the recognition end sequences may further comprise an adaptor portion. In some embodiments, the transposition results in fragmented target DNA, wherein at least some of the fragments comprise a different adaptor and recognition end sequence on each end, for example, as depicted in the top panel of FIG. 2. In some embodiments, tagged DNA libraries suitable for high-throughput sequencing are created by combining di-tagged DNA fragments with amplification primers and DNA polymerase under conditions suitable for primer hybridization and PCR amplification to occur, for example, as depicted in the middle and bottom panels of FIG. 2.

[0036] The disclosed compositions, methods, and kits may comprise any naturally available transposase or genetically engineered transposase capable of binding with the disclosed oligonucleotides to form a transpososome capable of in vitro transposition.

[0037] Certain disclosed compositions, methods, and kits comprise a first oligonucleotide comprising a first recognition end sequence and a second oligonucleotide comprising a second recognition end sequence, where the first recognition end sequence and the second recognition end sequence are different. In some embodiments, a first oligonucleotide, a second

oligonucleotide or both, further comprise an adaptor portion that is upstream of the recognition end sequence. In some embodiments, the first oligo comprises a first recognition end sequence and a first adaptor sequence and the second oligo comprises a second recognition end sequence and a second adaptor sequence, wherein the first recognition end sequence and the first adaptor are different from the second recognition end sequence and the second adaptor.

[0038] Certain composition, method and kit embodiments further comprise a third oligonucleotide comprising a third recognition end sequence and a fourth oligonucleotide comprising a fourth recognition end sequence, where the third and fourth recognition end sequences. In some embodiments, a third oligonucleotide, a fourth oligonucleotide or both, further comprise an adaptor portion that is upstream of the recognition end sequence. In some embodiments, the third oligonucleotide comprises a third recognition end sequence and a third adaptor sequence and the fourth oligonucleotide comprises a fourth recognition end sequence and a fourth adaptor sequence, wherein the third recognition end sequence and the third adaptor are different from the fourth recognition end sequence and the fourth adaptor. In some embodiments the first and third adaptor sequences are the same or different and the second and fourth adaptor sequences are the same or different. In certain embodiments, the fragments in the DNA library comprise (a) a first subpopulation of fragments comprising a first recognition end sequence on one end and a second recognition end sequence on the other end and (b) a second subpopulation of fragments comprising a third recognition end sequence on one end and a fourth second recognition end sequence on the other end. In some embodiments, the number of di- tagged fragments in the first subpopulation and the second subpopulation are approximately equal.

[0039] The recognition end sequence can be a naturally occurring sequence for the transposase of choice, or it can be an engineered sequence designed for a particular transposase and providing additional or alternative functions for the oligonucleotide or DNA fragment where it is incorporated or transposed.

[0040] In certain exemplary embodiments, the recognition end sequence comprise: 5'- AGATCTGATCAAGAGACAG-3 ^* (SEQ ID NO: l); 5 ^* - CTGTCTCTTGATCAGATCT-3 ^* (SEQ ID NO:2); 5 ^* - ACTTGTGTATAAGAGTCAG-3 ^* (SEQ ID NO: 3); 5'-

CTGACTCTTATAC AC AAGT-3 ' (SEQ ID NO:4). In some embodiments, the first recognition end sequence and the second recognition end sequence are double stranded DNA sequence comprising a sequence for one strand of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. Certain embodiments comprise: (1) a first composition comprising a first

oligonucleotide comprising a first recognition end sequence comprising SEQ ID NO: l, a second oligonucleotide comprising a second recognition end sequence comprising SED ID NO:2 and a transposase; and (2) a second composition comprising a third oligonucleotide comprising a third recognition end sequence comprising SEQ ID NO:3, a fourth oligonucleotide comprising a fourth recognition end sequence comprising SED ID NO:4 and a transposase. In some embodiments, such first and second compositions are incubated under conditions suitable for transposome formation to occur. In some embodiments, the first and/or the second compositions comprising transpososome complexes are combined with target DNA. In some embodiments, the first composition, the second composition, and the target DNA are combined simultaneously or the components are combined in any order. The combination is incubated under conditions suitable for transposition reactions to occur, resulting in fragmentation of the target with each fragment comprising a different end sequence than the corresponding end of the fragment. In some embodiments, where the oligonucleotides comprising the recognition end sequences further comprise adaptor sequences, transposition results in DNA fragments with ends comprising different end sequences and adaptors (for example, as depicted in FIG. 2).

[0041] In some embodiments, the first oligonucleotide, the second oligonucleotide, the transposase, and the target DNA are combined at the same time. In other embodiments, the first oligonucleotide, the second oligonucleotide, and the transposase are combined to make a complex prior to combining the complex with the target DNA. Any number of additional substances can be included in the disclosed compositions and methods. Those in the art will appreciate that the identity, number, and amount of the various additional components will typically be dictated by the application for the composition or the specific optimal activity requirements for a particular transposase employed.

[0042] In certain embodiments, DNA fragments comprising adaptors are combined with pairs of primers (for example but not limited to SEQ ID NO: 7 and SEQ ID NO: 8) under conditions suitable for primer hybridization. According to various embodiments, the primers are suitable for use as amplification or sequencing primers. In certain embodiments, the primers used for PCR amplification include 3' regions comprising sequences that are part of the recognition end sequence(s). In some embodiments, primers include 5' sequences that do not include sequences that are part of the recognition end sequence(s). In some embodiments, one or both primers include sequences (sometimes referred to as tags) that allow amplicons generated using such primers to hybridize to a solid support for sequencing.

[0043] In some embodiments, the tags provide priming sites for sequencing using a DNA polymerase. In some embodiments, the tags also provide sites for capturing the fragments onto a surface, such as a bead (e.g., prior to emulsion PCR amplification). In some embodiments, the DNA fragment libraries used as templates for next generation sequencing comprise 5'- and 3'- tagged DNA fragments or "di-tagged DNA fragments," i.e., the fragments comprise a different recognition end sequence in the 5 ' portion of the fragment than the recognition end sequence that is found in the 3 ' portion of that DNA fragment.

Example 1

Formation of transpososomes (transposase-transposon complexes).

[0044] A first composition comprising lmicroliter (μΕ) lx TE buffer (lOmM Tris pH8.0, ImM EDTA), 5μί lOOuM first oligonucleotide comprising a first recognition end sequence (SEQ ID NO: l) and 5μί lOOuM second oligonucleotide comprising a second recognition end sequence (SEQ ID NO:2) was incubated at 90°C for 5 minutes. The temperature was decreased to 25° C, then maintained at 25°C for 2 hours.

[0045] A second composition comprising Ι μΕ lx TE buffer (lOmM Tris pH8.0, ImM EDTA), 5μΙ, lOOuM third oligonucleotide comprising a third recognition end sequence (SEQ ID NO:3) and 5μί lOOuM fourth oligonucleotide comprising a fourth recognition end sequence (SEQ ID NO:4) was incubated at 90°C for 5 minutes. The temperature was decreased to 25° C in a step-wise manner, then maintained at 25°C for 2 hours.

[0046] Transpososome 1 preparation. Nine lx TE buffer, ΙμΕ of the first

composition comprising the first and second oligonucleotides, and ΙΟμΙ, recombinant transposase Tn5 (2ug^L; obtained from a Tn5 gene mutated to prevent expression of the inhibitor protein and possess enhance the transposition activity) were combined and incubated at room temperature for 1 hour.

[0047] Transpososome 2 preparation. Nine μΐ, lx TE buffer, ΙμΕ of the second composition comprising the third and fourth oligonucleotides, and ΙΟμΙ _^ recombinant transposase Tn5 (2ug^L) were combined and incubated at room temperature for 1 hour. The two transposome preparations were mixed together, then stored at -20°C.

Example 2

Generation of tagged DNA fragment libraries from gDNA.

[0048] Fifty nanogram of four genomic DNAs (human genomic DNA 9948 (MCLAB), bacteriophage lambda DNA (Takara), Helicobacter pylori (H. pylori) DNA (Coriell) and E. coli DNA (MCLAB) were adjusted to final volumes of 10 μί using nuclease-free Η20 in four PCR tubes, one for each DNA target. To each tube was added 10 μί of 5x reaction buffer (45mM Tris Acetate pH7.5, 20mM Magnesium Acetate, 40mM Potassium Acetate, 20mM Potassium

Glutamate and 5% N,N-Dimethylformaide), 5 μΐ _^ of the mixture of transposome 1 and 2 (see Example 1), and 25 μΐ, nuclease-free H20, and the tubes were mixed gently. The tubes were then placed into a thermocycler (MJ Research PTC-200 Thermal Cycler) programmed to run: 55°C for 5 minutes, then hold at 10°C.

[0049] The resulting tagmented DNA was mixed with 180 μΐ _^ Binding Buffer (Zymo DNA Clean & Concentration Kit), transferred to a column (Zymo), then centrifuged at 10,000 rpm in an 5415C Centrifuge (Eppendorf) for 1 minute, then washed twice with 300 μΐ, Wash Buffer (Zymo). Fourteen μΕ of DNA Resuspension Buffer (Zymo) was added directly to the column matrix, the column was incubated 2 minutes at room temperature, then the elute was collected by centrifuging the column at 10,000 rpm for 1 minute.

[0050] The purified tagmented DNA was amplified via 9-cycle PCR program with addition of extra sequences: index 1 (i7) and index 2 (i5) (Nextera Index Kit, Illumina) for sequencing, as well as common adapters (P5 and P7; SEQ ID NO:7 and SEQ ID N0:8, respectively) for cluster generation and sequencing.

[0051] The PCR mixture included 25μΙ, Amplification MasterMix (MCLAB 1-5™ 2X High-Fidelity Master Mix) 5μΕ PCR PRimerMix (lOuM P5 and P7), 4μΕ each index 1 and index 2, and the tagmented DNA. The PCR program was: 1) 72°C 3 minutes, 2) 98°C 3 minutes, 3) 9 cycles of: 98°C 10 seconds, 65°C 30 seconds, 72°C 3 minutes, then 4) hold at 10°C.

[0052] Fifteen μΕ of each of the four PCR products were analyzed on a 2% agarose gel As seen in FIG. 4, the resulting amplification products were fragments of about 500 bp.

[0053] Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications may be made without departing from the teachings herein. The foregoing examples are provided to better illustrate the present teachings and are not intended to limit the scope of the teachings herein. Furthermore, various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Certain aspects of the present teachings may be further understood in light of the following claims.