METHODS AND SYSTEMS FOR MULTIPLEX GENE AMPLIFICATION FROM ULTRA-LOW DNA INPUT AMOUNTS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Ser. No. 62/729,921 , entitled“Multiplex Gene Sequencing From Ultra-Low DNA Input Amounts” to Scharfe et al. , filed September 11 , 2018, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Governmental support under Grant No. HD081355 awarded by the National Institute of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] This application hereby incorporates by reference the material of the electronic Sequence Listing filed concurrently herewith. The material in the electronic Sequence Listing is submitted as a text (.txt) file entitled“06034_Sequences_ST25.txt” created on August 1 , 2019, which has a file size of 876 KB, and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0004] The present invention is directed to polymerase chain reactions (PCR) and applications thereof, more particularly, multiplex PCR methods that allow for simultaneous amplification of multiple target sequences from ultra-low amounts of template nucleic acids.

BACKGROUND OF THE INVENTION

[0005] PCR is a commonly used method in biology and medicine for a number of purposes, including mutation detection, identification of individuals, diagnostic testing, genotyping, and nucleic acid sequencing. Current methods typically can only amplify a limited number of target sequences at a time and require large quantities of high-quality starting template (DNA or RNA). Unfortunately, many biological samples, including dried blood spots, possess low quantities of nucleic acids and can be of limited quality, which makes PCR on these samples difficult and nearly impossible for amplifying multiple targets simultaneously. As such, a need in the art exists to develop systems and methods that enable amplification of multiple target sequences in low quality and/or quantity DNA samples.

SUMMARY OF THE INVENTION

[0006] Systems and methods for multiplex nucleic acid amplification in accordance with embodiments of the invention are disclosed. In one embodiment, a composition for performing PCR includes a universal primer and a plurality of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer, wherein the forward and the reverse primer comprises a general 5’-A-B-3’ structure, where A represents the universal primer sequence and B represents a target specific sequence.

[0007] In a further embodiment, the universal primer possesses a melting temperature of approximately 69°C to approximately 72°C.

[0008] In another embodiment, the plurality of primer pairs is at least 50 primer pairs.

[0009] In a still further embodiment, the plurality of primer pairs is at least 500 primer pairs.

[0010] In still another embodiment, the forward primers and reverse primers further comprise a spacer sequence, C, wherein the forward and reverse primers comprise a general 5’-A-C-B-3’ structure.

[0011] In a yet further embodiment, the spacer in each forward primer consists of the sequence TCTG and the spacer in each reverse primer consists of the sequence AGAC.

[0012] In yet another embodiment, the universal primer and the plurality of primer pairs are at a ratio of 10: 1 .

[0013] In a further embodiment again, the universal primer sequence is SEQ ID: 2818.

[0014] In another embodiment again, the target specific sequence for each forward primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 940-1878, and each reverse primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 1879-2817. [0015] In a further additional embodiment, a method of targeted sequencing of an individual, includes the steps of amplifying a plurality of target sequences in a sample using a first PCR reaction to create amplicons containing a universal primer sequence, wherein the first PCR reaction contains a universal primer, a plurality of forward primers, and a plurality of primer pairs, wherein each primer pair comprises a forward primer and a reverse primer, wherein the forward and the reverse primer comprises a general 5’-A- B-3’ structure, where A represents the universal primer sequence and B represents a target specific sequence, generating a sequencing library from the amplicons using a second PCR reaction, wherein the second PCR reaction contains sequencing adapter primers comprising a general 5’-D-A-3’ structure, where D represents a sequencing adapter sequence and A represents the universal primer sequence, and sequencing the sequencing library on a sequencing platform.

[0016] In another additional embodiment, the method includes obtaining a sample.

[0017] In a still yet further embodiment, the sample is a dried blood spot.

[0018] In still yet another embodiment, the forward primers, reverse primers, and sequencing adapter primers further comprise a spacer sequence, C, wherein the forward and reverse primers comprise a general 5’-A-C-B-3’ structure and the sequencing adapter primers comprise a general 5’-D-A-C-3’ structure.

[0019] In a still further embodiment again, the universal primer possesses a melting temperature of approximately 69°C to approximately 72°C.

[0020] In still another embodiment again, the universal primer and the plurality of primer pairs are at a ratio of 10: 1 .

[0021] In a still further additional embodiment, the universal primer sequence is SEQ ID: 2818.

[0022] In still another additional embodiment, the plurality of primer pairs is at least 50 primer pairs.

[0023] In a yet further embodiment again, the plurality of primer pairs is at least 500 primer pairs. [0024] In yet another embodiment again, the target specific sequence for each forward primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 940-1878, and each reverse primer in the plurality of forward primers is selected from the group consisting of SEQ ID NOs: 1879-2817.

[0025] In a yet further additional embodiment, the sequencing adapter sequence is selected from the group consisting of SEQ ID NOs: 2819-2820.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings where:

[0027] Figure 1 illustrates a method for obtaining a sample and treating an individual in accordance with embodiments.

[0028] Figures 2A-2F illustrate primer sequences for a multiplex PCR reaction in accordance with embodiments.

[0029] Figures 3A-3B illustrate results of multiplex PCR reactions in accordance with embodiments.

[0030] Figures 4A-4D illustrate primer sequences for a PCR reaction to generate sequencing libraries in accordance with embodiments.

[0031] Figures 5A-5D illustrate primer sequences for a PCR reaction to generate sequencing libraries in accordance with embodiments.

[0032] Figures 6A-6C illustrate sequencing reaction primers in accordance with embodiments.

[0033] Figures 7A-7C illustrate sequencing reaction primers in accordance with embodiments.

[0034] Figures 8A-8B illustrate results of sequencing reactions in accordance with embodiments.

[0035] Figures 9A-9C illustrate sequence analysis results in accordance with embodiments. DETAILED DISCLOSURE OF THE INVENTION

[0036] The present disclosure may be understood by reference to the following detailed description, taken in conjunction with the drawings as described below. It is noted that, for purposes of illustrative clarity, certain elements in various drawings may not be drawn to scale.

[0037] In accordance with the provided disclosure and drawings, systems and methods of performing multiplex PCR on low and ultra-low quantities of starting template using custom primer sequences having a homotag. In some embodiments, these primers are capable of amplifying over 100 targets simultaneously and/or are capable of amplifying targets from low quantities of starting template. Along with these primers, sequencing methods are provided capable of sequencing the targets from numerous individuals, simultaneously. Additionally, methods for analyzing the sequencing results to advance treating an individual are provided.

[0038] Traditional genetic testing or screening typically assesses an individual’s genetics via hybridization, PCR, or sequencing. In hybridization panels, an individual’s DNA is hybridized to a panel of known variants or mutations to identify which variants the individual possesses. While these panels can typically screen for a large number of targets, the panels are limited in that they can only identify variants that have previously been described and/or identified and cannot identify novel or previously unknown variants and can be limited in the ability to detect structural variation.

[0039] Similarly, PCR-based methods are typically limited to known variants but also has a number of problems that arise when amplifying multiple targets within a single reaction, including the increased levels of primers needed for the amplification of each target. With the addition of each target sequence, two additional primers need to be added the reaction. Adding additional primers to a reaction increases the likelihood of forming primer dimers or off-target amplification in the reaction, thus inhibiting amplification of the correct target. One solution has been to add additional template nucleic acid (either DNA or RNA) to the sample to increase the likelihood that the primers will amplify the correct sequence. Another solution is to reduce the concentration of the primers, but this strategy suggests a reduction in PCR sensitivity. Current methods of multiplex PCR have only resulted in amplification of a limited number (e.g., less than about 20) of individual targets in a single reaction. Embodiments herein describe a methods and systems to amplify large numbers of target sequences, including amplifying greater than 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 targets in a single PCR reaction. Further embodiments are directed to methods of sequencing the amplified targets.

[0040] Additionally, some biological samples are limited in quantity and/or lack large quantities of DNA or RNA, thus limiting the ability for an individual to amplify multiple targets in a single reaction. For example, dried blood spots (DBSs) are regularly taken from babies shortly after birth. DBSs provide a chance to assess newborns for genetic health defects, disorders, or diseases at a very early time point, which may be important for early life care. Flowever, DBS samples contain only small and varying amounts of blood, thus the nucleic acid content within a DBS is limited. Adding multiple primer pairs to a reaction with limited input template would quickly overwhelm in the input template and increase the likelihood of primer dimers or other inhibiting structures.

[0041] Finally, sequencing is a great alternative by providing full sequence reads and identification of novel variants that could be missing from other panels. Flowever, in genetic testing, typically only a panel of genes or genetic elements are relevant, thus whole genome sequencing would reveal much additional data that may not have any effect on underlying diseases or conditions in an individual. Traditional targeted sequencing typically utilizes a combination of hybridization and PCR to isolate and amplify a subset of targets with added costs in reagents, labor, and equipment. Thus, there exists a need in the art for PCR-based panels to amplify a large number of sequences to reduce costs and improve genetic screening, especially with samples containing low amounts of nucleic acids.

[0042]An example of a targeted panel of genes is the Recommended Universal Screening Panel (RUSP), which can detect more than forty metabolic disorders that have historically caused significant morbidity and mortality in children. ( See American College of Medical Genetics Newborn Screening Expert G. Newborn screening: toward a uniform screening panel and system --executive summary. Pediatrics. 2006; 1 17(5 Pt 2):S296- 307; and Urv TK, Parisi MA. Newborn Screening: Beyond the Spot. Adv Exp Med Biol. 2017; 1031 :323-346; the disclosures of which are incorporated herein by reference in their entireties.) Flowever, typical RUSP assays in newborns uses tandem mass spectrometry (MS/MS). (See Carreiro-Lewandowski E. Newborn screening: an overview. Clin Lab Sci. 2002; 15(4):229-238; Chace DH, Kalas TA, Naylor EW. Use of tandem mass spectrometry for multianalyte screening of dried blood specimens from newborns. Clinical chemistry. 2003;49(1 1 ): 1797-1817; and Turgeon C, Magera MJ, Allard P, et al. Combined newborn screening for succinylacetone, amino acids, and acylcarnitines in dried blood spots. Clinical chemistry. 2008;54(4):657-664; the disclosures of which are incorporated herein by reference in their entireties.) While beneficial in most respects, MS/MS screening is tuned to maximize the number of newborns identified, with sensitivity favored over specificity. This approach increases the number of false-positive results, leading to considerable emotional and financial burdens of follow-up testing, unneeded medical precautions for false-positive cases and diagnostic delays for some infants. ( See Waisbren SE, et al. Effect of expanded newborn screening for biochemical genetic disorders on child outcomes and parental stress. JAMA. 2003;290(19):2564-2572; the disclosure of which is incorporated herein by reference in its entirety.) To reduce the number of false-positive cases without compromising sensitivity, screen-positive results are followed by second-tier testing at higher specificity. ( See Matern D, et al. Reduction of the false-positive rate in newborn screening by implementation of MS/MS-based second-tier tests: the Mayo Clinic experience (2004-2007). Journal of inherited metabolic disease. 2007;30(4):585-592; the disclosure of which is incorporated herein by reference in its entirety.) As such, second-tier tests measure more specific disease markers (e.g., organic acids) to confirm (true positive) or reject (false positive) the primary screen result. Second-tier tests are typically not part of the primary screen due to assay complexity, limited throughput, analysis time and cost. ( See e.g., Chace DH, Hannon WH. Impact of second-tier testing on the effectiveness of newborn screening. Clinical chemistry. 2010;56(1 1 ): 1653-1655; the disclosure of which is incorporated herein by reference in its entirety.) However, both primary and secondary screening utilizes the original newborn DBS to avoid a new blood draw and minimize turnaround time.

[0043] The advent of rapid, inexpensive next-generation sequencing (NGS) promises to revolutionize newborn screening. ( See e.g., Berg JS, et al. Newborn Sequencing in Genomic Medicine and Public Health. Pediatrics. 2017; 139(2); the disclosure of which is incorporated herein by reference in its entirety.) Incorporating NGS-based analysis at the earliest stage in the screening process could drastically streamline the diagnostic work- up following an abnormal NBS result, but has several challenges. Previous studies using residual DBS for NGS either required large amounts of DBS material, or used whole- genome amplification for sequence library preparation. ( See Hollegaard MV, et al. Archived neonatal dried blood spot samples can be used for accurate whole genome and exome-targeted next-generation sequencing. Molecular genetics and metabolism. 2013; 1 10(1 -2):65-72; Bhattacharjee A, et al. Development of DNA confirmatory and high- risk diagnostic testing for newborns using targeted next-generation DNA sequencing. Genetics in Medicine: official journal of the American College of Medical Genetics. 2015; 17(5):337-347; Cantarel BL, et al. Analysis of archived residual newborn screening blood spots after whole genome amplification. BMC genomics. 2015; 16:602; and Poulsen JB, et al. High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA. PLoS One. 2016; 1 1 (4):e0153253; the disclosures of which are incorporated herein by reference in their entireties.) A less expensive and more efficient approach is multiplex gene sequencing from DBS from a multiplex PCR reaction, using a panel of genes relevant to the specific disease(s) or biological condition(s) detected in primary newborn screening.

[0044] Current NGS diagnostics are suboptimal for NBS due to their inability to accommodate DBS-derived material. Newborn DBS samples contain only small and varying amounts of blood, from which multiple punches are taken for NBS for the various conditions on the panel. The small amount of dried blood remaining limits the amount of extractable DNA for use in second-tier testing. Previous studies using residual DBS for NGS either required large amounts of DBS material, or used whole-genome amplification for sequence library preparation. ( See Hollegaard MV, et al. Archived neonatal dried blood spot samples can be used for accurate whole genome and exome-targeted next- generation sequencing. Molecular genetics and metabolism. 2013; 1 10(1 -2):65-72; Bhattacharjee A, et al. Development of DNA confirmatory and high-risk diagnostic testing for newborns using targeted next-generation DNA sequencing. Genetics in medicine: official journal of the American College of Medical Genetics. 2015; 17(5):337-347; Cantarel BL, et al. Analysis of archived residual newborn screening blood spots after whole genome amplification. BMC genomics. 2015;16:602; and Poulsen JB, et al. High- Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA. PLoS One. 2016; 1 1 (4):e0153253; the disclosures of which are incorporated herein by reference in their entireties. A more efficient approach is multiplex gene sequencing from DBS, using a panel of genes relevant to the specific condition(s) detected in primary newborn screening, which is incorporated into numerous embodiments. Further

[0045] Turning to Figure 1 , a method 10 is illustrated that incorporates numerous described herein and how many of the embodiments relate into a larger process. In particular, at Step 12, a sample is obtained. In numerous embodiments, the sample contains nucleic acids (e.g., DNA or RNA). In many embodiments, the sample is a blood sample. In some embodiments, the sample is a DBS. It should be noted that many different samples are known in the art.

[0046] At Step 14, DNA is isolated from the sample in numerous embodiments. The DNA may be isolated by any means known in the art that is sufficient for the specific tissue and/or source of the sample. Many embodiments will isolate DNA from a DBS using methods designed to yield the maximum quantity of nucleic acids possible. Methods for isolating DNA from DBSs according to many embodiments are described further in depth below.

[0047] At Step 16, a PCR reaction is performed in many embodiments. For some embodiments, the PCR reaction amplifies a single amplicon from the template nucleic acids isolated from the sample. In many embodiments, multiplex PCR reactions are performed to isolate many targets simultaneously. Additional embodiments will utilize unique sequences concatenated to target specific primers to increase amplicon efficiency. Additional details on primer design will be described in detail below.

[0048] At Step 18 of many embodiments, a sequencing library or target sequences is generated. In a number of embodiments, the sequencing library is generated using PCR. A sequence library in accordance with embodiments will add specific nucleic acid sequences to allow the target amplicons to be sequenced, such as adapter and index sequences. Numerous embodiments will append lllumina adapters to the amplicons generated from the PCR reaction of Step 16. [0049] The library of target sequences will be sequenced at Step 20 of many embodiments. Many methods and platforms for sequencing nucleic acids are known in the art, many of which will be sufficient for sequencing libraries generated in embodiments herein. However, a number of embodiments will utilize an lllumina platform, such as a MiSeq, HiSeq, HiScan, iSeq, MiniSeq, NextSeq, NovaSeq, and/or any other lllumina platform.

[0050] Variants will be identified and annotated in the sequence of many embodiments at Step 22 of many embodiments. Numerous methods exist in the art for identifying variants, including GATK, Annovar, and many other available software packages and/or resources.

[0051]At Step 24, many embodiments will treat an individual based on the identified and annotated variants. In many embodiments, the treatments are known in the art for an affliction, condition, and/or disease identified at Step 22.

[0052] While the above method 10 contains a number of steps, not all steps are necessary to be performed in all embodiments. Additionally, method 10 is meant to illustrate a number of embodiments that stand alone as separate embodiments, which can be integrated into larger processes, methods, systems, kits, etc. Additionally, numerous embodiments may be able to perform some steps simultaneously, nearly simultaneously, and/or in an order that differs from what is illustrated in Figure 1 .

DNA Isolation

[0053] Many embodiments are directed to amplifying target sequences using DBS samples collected from individuals, including newborn babies. As noted above, DBS samples contain limited and varying quantities of DNA. As such, many embodiments isolate DNA from DBS in a method to maximize DNA yield. In some embodiments, one or more punches are taken from a DBS. In several embodiments, a single 3mm punch is taken from a DBS from one individual. In various embodiments, the punch(es) are washed one or more times with 10mM NaOH. In numerous embodiments, the punch(es) are suspended in a volume of 10mM NaOH and heated to allow DNA to elute from the DBS. In various embodiments, the punch(es) is suspended in 50pl_ of 10mM. In certain embodiments, the punch(es) are heated for a period of 5, 10, 15, 20, or 30 minutes at 99°C. Various embodiments will mix and/or transfer the liquid, which contains isolated DNA, to a fresh tube for further processing.

[0054] Many embodiments will obtain samples from multiple individuals simultaneously. For example, punches can be taken from 96, 192, or 384 individuals simultaneously to allow DNA isolation using 96-well, 192-well, or 384-well plates.

Multiplex PCR Reactions

[0055] Many embodiments are directed to components and methods for performing PCR reactions in accordance with many embodiments is described. Turning to Figures 2A- 2B, forward 102 and reverse 104 PCR primers in accordance with many embodiments are illustrated. The primers 102, 104 start at the 5’-end 106 to the 3’-end 108 of each primer 102, 104. As with many PCR reactions, forward 1 10 and reverse 1 12 target specific primers are included in order to frame the target sequence and allow amplification of the target sequence from template nucleic acids. The amplified target molecules are referred to as amplicons. Additionally, homotags 1 14, 1 16 are attached at the 5’-ends 106 of the forward 102 and reverse 104 primers. A homotag in accordance with many embodiments is a universal primer that allows amplification of the amplicons independent of the target sequence. In many embodiments, the homotags 1 14, 1 16 that are part of the PCR primers 102, 104 possess the same sequence, thus allowing a single-primer amplification of target amplicons. In general, the structure of primers 102, 104 of many embodiments can be described as 5’-A-B-3’, where A is a homotag 1 14, 1 16, and B is target specific primer 1 10, 1 12.

[0056] In a number of embodiments, the PCR primers 102, 104 will be designed to avoid aberrant amplification, off-target amplification, and/or other issues that may arise because of poor primer design. When designing PCR primers 102, 104, a number of methods can be utilized, including automation with available software packages. In several embodiments, target sequences, such as entire genes, regions, and/or other significant areas, will be analyzed to avoid problematic sequences, such as repetitive elements. Many of these embodiments will utilize repeat masking software, such as RepeatMasker to block off repetitive elements within these regions. For example, if an entire gene sequence is identified as a target sequence, the target sequence may include introns, exons, 5’-UTRs, 3’UTRs, in addition to other genetic elements. Some of these features can include repetitive sequences that can interfere with PCR if used as a target specific primer 1 10, 1 12. By masking these sequences, these regions will not be selected as target specific primers 1 10, 1 12. Once certain elements are masked, target specific primer 1 10, 1 12 will be designed in many embodiments. The primer design can be performed manually or automated using programs such as Primer3. When multiplexing PCR reactions, the target specific primers 1 10, 1 12 are designed to have similar characteristics, such as size, melting temperature, GC content, amplicon size of the resulting amplicon, and any combination thereof. In some embodiments, target specific primers 1 10, 1 12 will have an average size of approximately 20-30 base pairs (bp), and amplicon size of approximately 300-500bp. In several embodiments, the target specific primers 1 10, 1 12 will range in size from 21 -27bp and have an average size of 23bp and amplify targets ranging from 350-500bp with an average size of 412bp.

[0057] Once designed, the entire sequence of PCR primer 102, 104 can be established, including homotags 1 14, 1 16. Once fully designed, additional quality control metrics will be performed in a number of embodiments. For example, sequences for PCR primers 102, 104 can be assessed for primer-dimer formation that can interfere with PCR reactions. Methods for optimizing primer design include the AutoDimer software package to assess secondary structure and/or primer dimer formation within a selection of PCR primer. If primers are predicted to form primer dimers or other interfering structures, the target specific primer sequences 1 10, 1 12 can be adjusted and reassessed until primer dimers or other structures are minimized.

[0058] In many embodiments, a pool of PCR primers 102, 104 are added in a reaction, where the target specific primers 1 10, 1 12 differ for the various targets, but the homotags 1 14, 1 16 remain the same. In several embodiments, the PCR primers 102, 104 are tested and optimized to reduce amplicon dropout and/or non-specific amplification. This optimization can include rebalancing a pool of primers (raising or lowering the concentration of specific primer pairs) or by altering the characteristics of the target specific primers 1 10, 1 12. It should be noted that one of skill in the art is capable of identifying issues with PCR primers, including dropout and/or non-specific amplification, and would know how to rebalance and/or altering primers within a reaction. In a number of embodiments, primer pairs that amplicons with low GC content will be increased, while primer pairs that amplify amplicons with high GC content will be reduced.

[0059] In certain embodiments, the homotags 1 14, 1 16 are designed to have a different (e.g., higher or lower) melting temperature than the target specific primers 106, 108. By altering melting temperature of the homotags 1 14, 1 16, aberrant amplification is less likely to occur from the presence of the homotags 1 14, 1 16. Additional embodiments will design homotags 1 14, 1 16 to lack homology with sequences within the genome sequence of a sample to be amplified. Lacking homology with the sample’s genome will aid in preventing aberrant or erroneous amplification. Many methods exist for determining which sequences possess or lack homology, including performing alignments of a particular sequence to the sample’s reference genome sequence (e.g., using BLAT, BLAST, and/or any other alignment software) or querying a K-mer database. It should also be understood that lacking homology does necessarily not mean possessing no homology with a reference sequence but lacking sufficient homology to prevent amplification under particular PCR reaction conditions.

[0060] In numerous embodiments, the homotags 1 14, 1 16 will have a higher melting temperature. Having a higher melting temperature for the homotags 1 14, 1 16 will allow for amplification of all amplicons using homotag-specific primers without allowing the target specific primers 1 10, 1 12 to anneal to template nucleic acids. In such a circumstance, amplicon amplification will occur rather than template amplification— i.e. , amplification will be based on amplicons containing homotag sequences rather than generating new amplicons from sample template. It should be noted that because nucleic acid amplification is directional from 5’ to 3’, homotag-specific primers will have the same sequence as the homotags 1 14, 1 16, such as illustrated in Figures 2E-2F, where the homotag-specific primers are only the homotag sequences 1 14, 1 16.

[0061] Turning to Figures 2C-2D, a number of embodiments will include forward 1 18 and reverse 120 spacer sequences between homotags 1 14, 1 16 and target specific primers 1 10, 1 12. Spacer sequences 1 18, 120 can be used to provide directionality into PCR primers 102’, 104’. Spacer sequences 1 18, 120 can be of any length to allow differentiation in direction. In a number of embodiments, the spacer sequences are 4-8bp long. In certain embodiments, the spacer sequences are 4bp sequences. In embodiments including spacers, the primers 102’, 104’ can be described to have a general structure of 5’-A-C-B-3’, where A is a homotag 1 14, 1 16, B is a target specific primer 1 10, 1 12, and C is a spacer 1 18, 120.

[0062] The RUSP panel contains 60 conditions including 34 core conditions and 26 secondary conditions. In a number of embodiments, the targets are selected based on the RUSP panel. In some embodiments, a panel of 72 genes are selected that include 64 genes associated with 46 different RUSP metabolic disorders and cystic fibrosis and an additional 8 genes associated with 7 metabolic disorders that are not currently in the RUSP metabolic disorders. Table 1 provides a list of conditions selected in some embodiments for use in many embodiments. The list in Table 1 includes conditions currently in the RUSP panel as well as additional conditions that will be selected in certain embodiments. In particular Table 1 identifies specific conditions in a RUSP panel by its ACMG code, the specific condition identified by that code, whether it is a core or secondary condition (if the condition is in the RUSP panel), condition type, genes and NCBI numbers for those genes that are associated with the condition, the current methodologies for determining the condition and the primary analyte for the analysis. Additional information regarding metabolic conditions can be found at www.hrsa.gov/advisory-committees/heritable-disorders/rusp/in dex.html; the disclosure of which is incorporated by reference in its entirety.

[0063] In a number of embodiments targeting the 72 genes as described in Table 2, the specific segments of these genes are selected as target amplicons. In many embodiments, the target amplicons are selected as all exons, flanking intronic regions, and key non-coding regions. In certain embodiments, the targeted amplicons are selected from the group consisting of SEQ ID NOs: 1 -939. In a number of embodiments, forward target specific primers 1 10 are selected from SEQ ID NOs: 940-1878 and reverse target specific primers 1 12 are selected from SEQ ID NOs: 1879-2817. Table 2 lists specific target names and identifies SEQ ID NOs for the target sequence and correlating forward and reverse target specific primers, where the forward primer SEQ ID NO and the reverse primer SEQ ID NO in a row form a primer pair for the target sequence SEQ ID NO in that same row. For example, SEQ ID NO: 940 and SEQ ID NO: 1879 form a primer pair for SEQ ID NO: 1 . It should be noted that the specific sequence for any of the target sequences SEQ ID NOs: 1 -939 are only representative of the specific target to be amplified. In many embodiments, variants will exist within the target amplicon for a particular sample, thus one primer pair will amplify a target sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a particular target sequence.

[0064] In certain embodiments, the homotag is SEQ ID NO: 2818. In some embodiments including spacers 1 18 and/or 120, the spacers are selected from the 5’-TCTG-3’ and 5’- AGAC-3’. In several embodiments of directional PCR primers 102’, 104’ (e.g., primers including spacers 1 18, 120) the forward spacer is 5’-TCTG-3’, and the reverse spacer is 5’-AGAC-3’. Thus, In many embodiments, the forward PCR primer 102 has a general structure of 5’-X-Y-Z-3’, where X is SEQ ID NO: 2818, Y is TCTG, and Z is any one of SEQ ID NOs: 940-1878, and reverse PCR primer 104 has a general structure of 5’-X-Q- R-3’, where X is SEQ ID NO: 2818, Q is AGAC, and R is any one of SEQ ID NOs: 1879- 2817. Numerous embodiments will pool multiple versions of PCR primers 102 and 104 or directional PCR primers 102’ and 104’.

[0065] An advantage having homotags 1 14, 1 16 with higher melting temperatures is that it will allow for a single reaction set up, which includes PCR primers 102, 104 or directional PCR primers 102’, 104’ along with homotag-specific primers 1 14, 1 16 within the same reaction tube or vessel. In such a circumstance, the reaction will comprise template nucleic acid (e.g., DNA and/or RNA), buffer, water, one or more forward primers, one or more reverse primer, a homotag-specific primer, nucleotide triphosphates (e.g., dNTPs and/or NTPs), a polymerase, and/or any other component known in the art to assist or promote PCR amplification. In many embodiments the pool PCR primers 102, 104 or directional PCR primers 102’, 104’ will be used at a total concentration of approximately 0.5mM (e.g., ±0.5mM), such that some embodiments will utilize 0.1 mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, or 1 .0mM of total PCR primers 102, 104 or directional PCR primers 102’, 104’. The homotag-specific primer 1 14, 1 16 will be placed in the reaction tube at a concentration equal to or greater than the concentration of pooled PCR primers 102, 104 or directional PCR primers 102’, 104’. As such, many embodiments will use a ratio of homotag-specific primers to PCR primers of 1 : 1 , 2: 1 , 3: 1 , 4: 1 , 5: 1 , 6: 1 , 7: 1 , 8: 1 , 9: 1 , 10: 1 or greater for the ratio of homotag-specific primers to PCR primers. Many embodiments will use a ratio of 10: 1 homotag-specific primers to PCR primers, such that approximately 5mM of homotag specific primers 1 14, 1 16 will be used in a reaction containing approximately 0.5mM of pooled PCR primers 102, 104 or directional PCR primers 102’, 104’.

[0066] Many embodiments are directed to DNA polymerization based on a DNA template, so these reactions will comprise a DNA template, buffer, at least one forward primer, at least one reverse primer, a homotag primer, dNTPs, and a polymerase. In many embodiments, the forward and reverse primers will possess a homotag sequence, such as those described herein. In many embodiments, the polymerase will be a DNA polymerase, such as Taq polymerase, while additional embodiments will utilize high fidelity polymerases, strand displacement polymerases, RNA polymerases, long-range polymerases, any other polymerase relevant to the type of reaction, and/or any combination thereof. Numerous embodiments will alter the temperature cycling of the reaction to include a first set of cycles with a lower annealing temperature to allow for template amplification followed by a second set of cycles with a higher annealing temperature to allow for amplicon amplification. For example, the first set of cycles will have an annealing temperature of approximately 50-69°C followed by a second set of cycles with an annealing temperature of approximately 70-72°C. Additional embodiments will include manipulations to the sets of cycles, such as ramping, touch-down, or any other methodology for amplification. Some specific embodiments utilize the following profile:

- 95°C for 3 minutes,

- 12 cycles of:

o 95°C for 16 seconds,

o 69-52°C for 2 minutes (reducing temperature by 1 5°C per cycle), o 72°C for 45 seconds,

- 10 cycles of:

o 95°C for 16 seconds,

o 72°C for 20 seconds,

o 72°C for 2 minutes. [0067] While the above is described in relation to a single sample, many embodiments will utilize common reaction plates to allow PCR amplification from multiple samples obtained from multiple individuals simultaneously. For example, 96 individual samples can be kept in a standard 96-well plate, which would allow for multiplex PCR reactions to be performed on all 96 samples simultaneously.

[0068] Turning to Figures 3A-3B, the success rates of amplifying 939 amplicons are illustrated in accordance with embodiments. Specifically, Figure 3A illustrates the number of sequencing reads per sample, which indicates the overall success rate of numerous embodiments of multiplex PCR reactions. As seen in Figure 3A, many samples were capable of producing approximately 1 million reads (e.g., 10 ⁶ reads). As illustrated, out of 78 samples, only 1 sample produced significantly fewer reads than other samples. Additionally, Figure 3B illustrates partial failure of multiplex PCR reactions as a function of uniformity, in accordance with many embodiments using DBS as the sample source. In particular, Figure 3B illustrates the percent of amplicons with at least 20% of the mean coverage of all amplicons. As illustrated in Figure 3B, only 2 samples out of 78 showed limited uniformity among the amplicons. In combination, Figures 3A-3B illustrate that many embodiments are capable of amplifying upwards of 900 amplicons from a very low amount of input nucleic acids.

Generating Sequencing Libraries

[0069] Many sequencing library generation methods are known in the art, including commercially prepared kits for building such libraries, such as those from KAPA, Ilium ina, and other companies. Flowever, many of these kits rely on ligating adapters to the ends of the target molecules rather than purely through PCR. Many embodiments will leverage the power of the homotags or homotags and spacer sequences to generate sequencing libraries for the target sequences.

[0070] Upon amplifying target sequences, a number of embodiments will generate sequencing libraries from the amplicons created during a PCR reaction, such as a PCR reaction described above. In certain embodiments, the sequencing libraries are created using a second PCR reaction. In a second reaction, additional primers can utilize the homotags to add additional adapters necessary for sequencing (e.g., Illumina P5 and/or P7 adapters). However, using the homotag sequences alone may not provide adequate representation of all amplicons in a reaction. For example, lllumina sequencing relies on different adapters residing at each end of a molecule to be sequenced. Using only the homotag sequences as primers would create an equal opportunity for the directional lllumina adapters to be added to each end, thus resulting in 50% of all molecules having the same sequencing adapter at each end of the molecule (P5-P5 or P7-P7). As such, many embodiments will utilize spacer sequences to create directionality for adding the sequencing adapters to the amplicons. As such, many adapters will have a structure as illustrated in Figures 4A-4B. In Figures 4A-4B, sequencing adapter primers 122, 124 utilize the homotag 1 14 and spacer sequences 1 18, 120 to amplify the amplicons. By using forward 1 18 and reverse 120 spacer sequences, attached at the 3’ -end 108 of the sequencing adapter primers 122, 124, the system can allow for only one sequencing adapter 126, 128 is added to each end of a molecule. In general, sequencing specific primers 122, 124 can be described to have the structure 5’-D-A-C-3’, where D is a sequencing adapter 126, 128, A is a homotag 1 14, 1 16, and C is a spacer sequence 1 18, 120. In many embodiments, the lllumina P5 adapter (SEQ ID NO: 2819) and lllumina P7 adapter (SEQ ID NO: 2820) will be used for the sequencing adapters 126, 128. In some embodiments, the forward sequencing adapter primer 122 will possess the structure 5’- lllumina P5 (SEQ ID NO: 2819)- homotag (SEQ ID NO: 2818)- forward spacer (TCTG)- 3’, and the reverse sequencing adapter primer 124 will possess the structure 5’-lllumina P7 (SEQ ID NO: 2820)-homotag (SEQ ID NO: 2818)-reverse spacer (AGAC)-3\

[0071] Further embodiments of sequencing adapter primers will include index sequences to allow for multiplex sequencing of multiple samples simultaneously. Many methods are known in the art to index and/or multiplex samples for sequencing. In a number of embodiments, a Nextera-style indexing system is used. Nextera indexing is a system that integrates one or two indexes onto the molecules in a sequencing library. By using two indexes, a specific combination of indexes identifies a single sample. For example, a set of 8 indexes at a first location and a set of 12 indexes at a second location creates 96 unique combinations, thus allowing a total of 20 indexes to uniquely identify 96 individual samples, which can be used for embodiments that have performed PCR reactions on 96 individual samples. Many embodiments will utilize index sequences, as shown in Table 3: Table 3: List of Indexes

[0072] In many embodiments, the indexes shown in Table 3 are integrated between sequencing adapters and the homotags, such as illustrated in Figures 4C-4D. In Figures 4C-4D, sequencing adapter primers 122’, 124’ are illustrated. Specifically, Figure 4C illustrates a first index 130 integrated between sequencing adapters 1 126 and homotag 1 14, while Figure 4D illustrates a second index 132 integrated between sequencing adapter 2 128 and homotag 1 14. In general, sequencing specific primers 122’, 124’ can be described to have the structure 5’-D-E-A-C-3’, where D is a sequencing adapter 126, 128, E is an index sequence 130, 132, A is a homotag 1 14, 1 16, and C is a spacer sequence 1 18, 120. As such, in some embodiments, the forward sequencing adapter primer 122’ will possess the structure 5’-lllumina P5 (SEQ ID NO: 2819)-first index (Table 3)-homotag (SEQ ID NO: 2818)-forward spacer (TCTG), and the reverse sequencing adapter primer 124’ will possess the structure 5’-lllumina P7 (SEQ ID NO: 2820)-second index (Table 3)-homotag (SEQ ID NO: 2818)-reverse spacer (AGAC)-3’.

[0073] Flowever, many embodiments will utilize custom sequencing read primers based on the homotags 1 14, 1 16; in some of these embodiments customs sequencing read primers will incorporate homotags 1 14, 1 16 and spacers 1 18, 120. In such embodiments, additional base pairs may be necessary to raise the melting temperature of the sequencing read primers. Figure 5A illustrates additional sequencing adapter primers 122”, 124” that include modifying spacers 134, 136 between sequencing adapters 126, 128 and homotags 1 14, 1 16. Additionally, Figure 5B illustrates sequencing adapter primers 122’”, 124’” that include modifying spacers 134, 136 between indexes 130, 132 and homotags 1 14, 1 16. In general, the sequencing adapter primers 122”, 124” can be described to have the structure 5’-D-F-A-C-3’, while sequencing adapter primers 122”’, 124”’ can be described to have the structure 5’-D-E-F-A-C-3’. In these embodiments, D is a sequencing adapter 126, 128, E is an index sequence 130, 132, F is a modifying spacer 134, 136, A is a homotag 1 14, 1 16, and C is a spacer sequence 1 18, 120.

[0074] In many embodiments, the first modifying spacer 134 will be a dinucleotide GG increase the melting temperature of a first sequencing read primer. Additional embodiments will utilize the oligonucleotide CCGTTTA as the second modifying spacer 136 to increase the melting temperature of a second sequencing read primer. As such, some embodiments of the forward sequencing adapter 122” will possess the structure 5’- lllumina P5 (SEQ ID NO: 2819)-first modifying spacer (GG)-homotag (SEQ ID NO: 2818)- forward spacer (TCTG)-3’, and the forward sequencing adapter primer 122”’ will possess the structure 5’-lllumina P5 (SEQ ID NO: 2819)-first index(Table 3)- first modifying spacer (GG)-homotag (SEQ ID NO: 2818)-forward spacer (TCTG)-3’. Similarly, some embodiments of the reverse sequencing adapter primer 124” will possess the structure 5’-lllumina P7 (SEQ ID NO: 2820)-second modifying spacer (CCGTTTA)-homotag (SEQ ID NO: 2818)-reverse spacer (AGAC)-3’, and reverse sequencing adapter primer 124”’ will possess the structure 5’-lllumina P7 (SEQ ID NO: 2820)-second index (Table 3)- second modifying spacer (CCGTTTA)-homotag (SEQ ID NO: 2818)-reverse spacer (AGAC)-3\

[0075] Many possible PCR cycling conditions can be used to create the sequencing libraries, based on the enzymes used, the melting temperature of the primers, and the length of the target molecules. In several embodiments the following cycling conditions are used:

- 98°C for 16 seconds,

- 13 cycles of:

o 98°C for 16 seconds,

o 72°C for 20 seconds. [0076] In a number of embodiments, the sequencing libraries are cleaned and/or purified after generation. In some embodiments, the cleaning uses commercially available kits, including kits using beads and/or columns. Some embodiments will use AMPure XP beads with a beat to sample ratio of 0.65: 1 , after which the sequencing libraries are eluted in 50pL of water.

[0077] Further embodiments will size select the library to eliminate too long or too short fragments, which could be generated from primer dimers and/or off target amplification. Many kits exist to perform such size selection, which can be used in embodiments. Some embodiments will utilize a Pippin Prep system for size selection.

[0078] Additional embodiments will also quantify the library, which can be accomplished with many means, including UV-Vis spectroscopy, fluorescence, qPCR, and/or electrophoresis. Certain embodiments will perform library quantification using a Bioanalyzer.

Sequencing Targets

[0079] As noted above, many possible sequencing platforms can be utilized to sequence targets generated in many embodiments. Also, many embodiments will utilize an lllumina platform to sequence the targets, including an lllumina MiSeq. Sequencing can be performed in any capacity allowed by a particular piece of equipment, including single read, paired-end reads, and/or mate-pair reads. Many embodiments will utilize paired- end read capacity of the platform in order to obtain as much sequence as possible for a particular target, including the entirety of the target sequence.

[0080] With such a configuration as illustrated in Figures 6A-6C, custom sequencing read primers can utilize the homotags and spacer sequences as custom sequencing read primers. Specifically, Figure 6A illustrates a first read sequencing primer 138; Figure 6B illustrates a second read sequencing primer 140 in accordance with many embodiments; and Figure 6C illustrates an indexing read primer 142, for embodiments including an index. In these embodiments, the sequencing read primers 138, 140 comprise a homotag 1 14, 1 16 located at the 5’-end of a spacer sequence 1 18, 120. In this configuration, the sequencing primers 138, 140 assure directionality of sequencing reads. In general, the sequencing read primers can be described to have the structure 5’-A-C-3’, where A is a homotag 1 14, 1 16, and C is a spacer sequence 1 18, 120. In embodiments which include Nextera-style indexes, a separate indexing read is necessary to read a second index. In these embodiments, the indexing read primer 142 is typically a reverse complement of second read sequencing primer 140. This configuration is illustrated in Figure 6C, which illustrates a reverse complement of reverse spacer 120’ at the 5’-end of reverse complement of homotag 1 16’. Thus, in general, indexing read primer 142 can be described to have the structure 5’-C’-A’-3’, where C’ is the reverse complement of a reverse spacer 120’, and A’ is the reverse complement of a homotag 1 16’. In many embodiments, first sequencing read primer 138 will have the sequence of SEQ ID NO: 2821 , which represents the structure 5’-homotag (SEQ ID NO: 2818)-forward spacer (TCTG)-3’, and second sequencing read primer 140 will have the sequence of SEQ ID NO: 2822, which represents the structure 5’-homotag (SEQ ID NO: 2818)-reverse spacer (AGAC)-3’. Also, when an indexing read primer 142 is used, it will have the sequence of SEQ ID NO: 2823, which represents the reverse complement of 5’-homotag (SEQ ID NO: 2818)-reverse spacer (AGAC)-3’.

[0081] As noted above, some embodiments will include modifying spacers 136, 138 to increase the melting temperature of the sequencing primers. Figures 7A-7C illustrate embodiments which include modifying spacers 136, 138 to increase the melting temperature. In particular, Figures 7A-7B illustrate sequencing read primers 138’, 140’ comprising a modifying spacer 136, 138 located at the 5’-end of a homotag 1 14, 1 16, which is located at the 5’-end of a spacer sequence 1 18, 120. In this configuration, the sequencing primers 138, 140 assure directionality of sequencing reads and an increased melting temperature to comply with machine settings, uniformity, or any other relevant factor for ensuring proper sequencing reads. In general, the sequencing read primers can be described to have the structure 5’-F-A-C-3’, where F is a modifying spacer 136, 138, A is a homotag 1 14, 1 16, and C is a spacer sequence 1 18, 120. In embodiments which include Nextera-style indexes, a separate indexing read is necessary to read a second index. In these embodiments, the indexing read primer 142’ is typically a reverse complement of second read sequencing primer 140’. This configuration is illustrated in Figure 7C, which illustrates a reverse complement of reverse spacer 120’ at the 5’ -end of reverse complement of homotag 1 16’, which is located at the 5’-end of reverse complement of a second modifying spacer. Thus, in general, indexing read primer 142’ can be described to have the structure 5’-C’-A’-F’-3’, where C’ is the reverse complement of a reverse spacer 120’, A’ is the reverse complement of a homotag 1 16’, and F’ is the reverse complement of a second modifying spacer 136’. In many embodiments, first sequencing read primer 138’ will have the sequence of SEQ ID NO: 2824, which represents the structure 5’ -first modifying spacer (GG)-homotag (SEQ ID NO: 2818)- forward spacer (TCTG)-3’, and second sequencing read primer 140’ will have the sequence of SEQ ID NO: 2825, which represents the structure 5’-second modifying spacer (CCGTTTA)-homotag (SEQ ID NO: 2818)-reverse spacer (AGAC)-3’. Also, when an indexing read primer 142’ is used, it will have the sequence of SEQ ID NO: 2826, which represents the reverse complement of 5’ -second modifying spacer (CCGTTTA)- homotag (SEQ ID NO: 2818)-reverse spacer (AGAC)-3’.

[0082] The raw data from a sequencer can be handled with innate software within the sequencing platform to generate the sequence files, including de-multiplexed sequence files (where multiple samples were multiplexed in the sequencing run). For example, MiSeq Control Software and MiSeq Reporter can analyze the raw image data and de- multiplexing of a run during and/or after a sequencing run has come to completion. Many embodiments will output the sequence in FASTA and/or FASTQ files for further analysis.

[0083] Turning to Figures 8A-8B, the success of sequencing libraries generated from embodiments are illustrated. In particular, Figure 8A illustrates that embodiments produce consistent read depths across multiple sequencing runs. Additionally, Figure 8B illustrates the success in sequencing individual samples, where the percent of base pairs with greater than 20x coverage are illustrated. As seen in Figure 8B, most samples tested produced at least 20x coverage in greater than 90% of all bases sequenced, and only 1 sample (out of 78 samples) produced lower read depth.

Identify and Annotate Variants

[0084] Once sequences have been generated, as above, the target sequences can be analyzed to identify variants and/or possible genetic conditions associated with these variants. In many embodiments, the sequences for each sample are aligned to a reference genome to identify particular variants. Alignment can be performed using any known software package for performing such alignments, including BLAST, BLAT, BWA, among others. Once sequencing reads are aligned, certain embodiments will identify variants using known software packages, including GATK or similar software packages. Variants in accordance with many embodiments including single nucleotide variants (SNVs), copy number variants (CNVs), and insertion-deletion variants (indels). Once variants are identified, a number of embodiments will annotate the variants for nomenclature and/or disease associations using databases of such information, including HGVS, OMIM, dbSNP, ClinVar, and ExAC. An example of such output can be seen in Table 4. Table 4 illustrates results from an embodiment identifying a sample ID (e.g., specific coordinate on sample plate), underlying genetic condition, and numerous categories identifying genes with multiple pathologic (P) and/or likely pathologic (LP) variants, genes with variants of unknown significance (VUS), and PubMed identifiers for known variants identified. Many embodiments will automate this process by pipelining all analysis beginning from the sequence reads to an output of relevant annotations for an individual.

[0085] Figures 9A-9C summarize variants identified in many embodiments. In particular, Figure 9A shows the distribution of the number of variants identified in control, false positive (MMA.FP) and true positive (MMA.TP) samples across 72 genes utilized in many embodiments. Similarly, Figure 9B illustrates the number of variants distribution of the number of variants identified in control, false positive (as identified via MS/MS) (MMA.FP) and true positive (MMA.TP) samples across 8 MMA genes utilized in some embodiments. Figure 9C illustrates the improvement in numerous embodiments, where pathogenic or likely pathogenic (P/LP) variants were identified in the true positive samples, thus reducing false positive rates, which reducing the demand for second-tier screening for many individuals, which in turn reduces the financial and/or emotional burden of receiving false positive results.

Treatment

[0086] In many embodiments, the results of sequencing and/or analysis, such as those described above will guide treatment of an individual. In certain embodiments, the results of the sequencing and/or analysis will be provided to a treating medical provider, such as a physician, nurse, or any other medical professional capable of providing treatment. Upon receiving such information as to metabolic conditions or other genetic diseases, the medical professional can utilize the information to select a treatment and provide the treatment to the individual. In many embodiments, the treatment step is an intervention, such as a drug, device, surgery, or other treatment designed to obviate symptoms and/or indications of the disease or condition, while additional embodiments will provide prophylaxis to the individual to prevent the onset of symptoms and/or complications that can arise due to the presence of a particular disease or condition identified from the sequencing and/or analysis.

EXEMPLARY EMBODIMENTS

[0087] Sequencing data supports the notion that embodiments described herein are capable of high plexity PCR amplification of target amplicons from very low nucleic acid inputs. The following data also details the ability to identify numerous metabolic conditions based on the presence of variants identified from target amplification. Accordingly, these data support the various embodiments of the invention as described.

Example 1 : Amplifying and Sequencing Optimizing Sulfhvdryl Blocking to Produce EVs

[0088] Background: DBS samples contain small and varying amounts of blood, thus contain very limited amounts of nucleic acids, including DNA. As such, analysis of metabolic diseases, through biochemical or chemical techniques or through genetic analysis is difficult.

[0089] Methods: In one exemplary embodiment, multiplex PCR and sequencing were performed for 939 amplicons starting from 80 DBS samples.

[0090] Study specimens: Research was approved by the Institutional Review Boards at Yale University (Protocol ID: 1505015917), Stanford University (Protocol ID: 30618) and the State of California Committee for the Protection of Human Subjects (Protocol ID: 13- 05-1236). De-identified residual DBS samples from 80 newborns from the California Biobank Program were used to validate the assay of this embodiment. These samples included 30 confirmed MMA cases, 30 MMA screen false-positives, and 20 DBS from healthy controls. In addition, metabolic data from a larger cohort of 803 newborns, consisting of 103 cases with confirmed MMA (24 mutO, 26 mut- 45 Cbl C, D, or F, 3 Cbl A or B, and 5 unclassified MMA), 502 screen false-positives, and 198 healthy controls were evaluated. All newborns had routine MS/MS metabolic screening performed through the California NBS program between 2005 and 2015. The 56 MS/MS analytes included free carnitine, acylcarnitines, amino acids and calculated ratios. Additional data collected included newborn race/ethnicity, gestational age (GA, in days), birth weight (in grams), total parenteral nutrition (yes or no), and newborn age at blood collection (in hours).

[0091] DNA Extraction: A single 3 mm punch was taken from each DBS using a PE Wallac instrument (Perkin Elmer, Santa Clara) and deposited into a 96-well plate. Three blank paper spots were punched between each sample to prevent cross-contamination. DBS punch spots were washed twice with 180 pL of 10 mM NaOH. Each punch spot was then suspended in 50 pL of 10 mM NaOH solution and heated at 99°C for 15 minutes in an Applied Biosystems GeneAmp PCR System 9700 (Life Technologies, Grand Island, NY). The supernatant, containing eluted DNA, was mixed by pipetting and then transferred to a clean tube containing 50 pL of 20 mM TrisCL pH 7.5. Two samples (D3, C1 1 in Table 4) of the 80 DBS failed in the DNA extraction.

[0092] Primer Design: A custom script integrating the primer design code from Primer 3 was used to generate target-specific forward and reverse primers for 939 amplicons for 362,013 base pairs (bp) of all exons and 20 bp of flanking intronic sequence of 72 genes based on hg19/GRCh37 (Table 2). Primer hybridization sites were selected to avoid common variants found in the National Center for Biotechnology Information (NCBI) single nucleotide polymorphism Database (dbSNP) build 137, June 2012 release. Primers were designed to have similar length (average 23 bp; range 21-27 bp), GC content, and amplicon size (average 412 bp, range 350-450 bp), matching the 2x250 bp paired-end sequencing chemistry on the MiSeq instrument (lllumina, San Diego, CA). Exons larger than 350 bp were covered by overlapping amplicons. Adapter sequences (e.g., homotag sequence SEQ ID NO: 2818) (24 bp) were included at the 5' end of each primer (e.g., SEQ ID NOs: 940-2817) for post-capture amplification.

[0093] Multiplex Target Capture: The 939 primer pairs (e.g., primers consisting SEQ ID NO: 2818 coupled to each of SEQ ID NOs: 940-2817) were pooled in one (1 ) tube for multiplex amplification 5 of 72 genes. Establishing a multiplex reaction in this embodiment required careful primer design and primer pool rebalancing, that included increasing or lowering the concentration of specific primers, replacing of failed primers, sequencing and data analysis. Primer optimization minimized amplicon dropout and non-specific amplification and achieved a 99% target base coverage from <10 ng of DBS DNA. Multiplex PCR was performed in a Veriti 96-well thermal cycler (Applied Biosystem, Foster City, CA) using 4-6 pL of extracted DNA in a 20 pl_ final volume and the KAPA2G Fast Multiplex PCR Kit (Kapa Biosystems, Wilmington, MA) across the following thermal profile: 95°C for 3 minutes, 12 cycles of 95°C for 16 seconds, 69-52°C (-1 .5°C per cycle) for 2 minutes, and 72°C for 45 seconds, followed by 10 cycles of 95°C for 16 seconds, 72°C for 20 seconds, and 72°C for 2 minutes. PCR cleanup was performed by adding 14 mI_ (0.7: 1 ) of AMPure XP beads (Beckman Coulter, Brea, CA) and clean up according to the manufacturer’s manual, with a final elution in 14 pL elution buffer.

[0094] Sequence Library Construction: 78 samples in four MiSeq runs were sequenced by multiplexing 17 to 22 samples per run. A no-template water control was included in each run. Sequencing library preparation was performed according to the manufacturer’s instructions (lllumina, San Diego, CA) using 5 pL of PCR product per sample. PCR was set up in 25 pL reactions, using common primers with sample specific indices and lllumina's P5 (SEQ ID NO: 2819) and P7 (SEQ ID NO: 2820) adapter sequences attached at the 5' end. Samples were barcoded with 8 bp dual indices (Table 3) according to lllumina's index sequencing protocol. KAPA2G Fast Multiplex PCR Kit (Kapa Biosystems, Wilmington, MA) was used to amplify DNA samples with the following cycling conditions: 98°C for 16 seconds, 13 cycles of 98°C for 16 seconds and 72°C for 20 seconds. Following DNA quantification for each sample, samples were pooled (approximately 200- 400 pL total volume) and purified using AMPure XP beads 5 (Beckman Coulter, Brea, CA) with a bead to sample ratio of 0.65:1 and eluted in 50 pL. 30 pL of the eluate was used for fragment size selection (440-720 bp) using the Pippin Prep system (Sage Science Inc., Beverly, MA), quantified the NGS library using Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA), and performed 2x250 bp PE sequencing on MiSeq (lllumina, San Diego, CA). [0095] Sequence Data Analysis: Image analysis and sample de-multiplexing was performed with the lllumina MiSeq Control Software version 2.4.1 and MiSeq Reporter version 2.5.1 .3 (lllumina, San Diego, CA). The resulting processed fastq files were aligned to the GRCh38 human reference genome using the Burrows-Wheeler Aligner (BWA- MEM, version 0.7.13- 126). Picard (version 2.8.1 ) was used to sort and convert files to BAM format. Quality control (QC) metrics were extracted for each sample from the BAM file, including total number of reads, percent reads that were properly paired and mapped to the reference genome, read depths for each amplicon, and read depth for individual base pairs within the target region (Figures 3A-3B and Figures 8A-8B). DNA variant calling was performed using GATK (version 3.6-0-g89b7209) with parameters as described previously. ( See e.g. , Lefterova Ml, et al. Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis. J Mol Diagn. 2016; 18(2):267-282; the disclosure of which is incorporated herein by reference in its entirety.) Annovar was used to annotate variants with the corresponding FIGVS DNA and protein level nomenclature in combination with public information relevant for variant annotation from OMIM, dbSNP, ClinVar and ExAC. For each sample (Table 3), sequence variants were classified as pathologic (P), likely pathologic (LP), likely benign, benign, or of unknown significance (VUS) based on ACMG standards and guidelines for interpretation of sequence variants.

[0096] Results: This embodiment shows the development of a highly multiplex PCR and NGS method for the analysis of 939 amplicons derived from 72 genes for inborn metabolic disorders from DBS. Out of 80 starting samples, only 2 samples failed the DNA extraction protocol. Figure 3A illustrates that only 1 of the remaining 78 samples failed in the multiplex PCR reaction, indicating the protocol is robust. Additionally, Figure 3B illustrates that only 2 samples had partially failed amplification as indicated by 2 standard deviations below the mean depth coverage of all amplicons, one of these samples also failed the total multiplex PCR amplification. Additionally, Figure 8A illustrates the robustness of embodiments, with consistent read depth across 4 sequencing runs on an lllumina MiSeq. Finally, Figure 8B illustrates the per-base coverage in all samples. Again, only one sample had significantly lower depth coverage, which is the same sample that failed total and partial amplification. In the end, 77 out of 80 DBS samples were able to proceed to full sequence analysis with greater than 90% of all bases having at least 20x coverage, which provides high confidence for variant calling.

[0097]Table 4 summarizes many of the results for the 77 sequenced samples that were successfully sequenced. These 77 samples include 28 MMA patients, of which 25 patients were identified with two variants in an MMA gene, while two patients (B2 and F4) had only one P/LP variant and one patient (F3) had no variant in the eight MMA genes analyzed. In the 29 MMA false-positive cases, two samples (E 10 and F110) were detected with two variants in an MMA gene, which in both samples were found in cis on the same amplicon reads and are thus located on the same chromosome. In the 20 control samples, two variants in an MMA gene were not detected. Analysis of the other 64 genes in this embodiment identified samples with two P/LP variants in PAH, PCCA, MTHFR, MLYCD, HPD, ACADVL, FAH, CPS1 , DBT, and NAGS. In two 15 samples (B8 and H6) the two P/LP were found on the same chromosome in MLYCD and PAH, respectively.

[0098] Conclusion: The results illustrate the ability to amplify very high amounts of amplicons from an ultra-low amount of starting DNA. From the multiplex PCR reaction, sequencing libraries can be constructed to identify variants and underlying metabolic and/or genetic conditions for an individual based on the low levels of starting DNA. While 2 samples failed the DNA extraction protocol, only 1 sample failed the amplification and sequencing reactions, emphasizing the power and robustness of embodiments.

DOCTRINE OF EQUIVALENTS

[0099] While the above description contains many specific embodiments of the invention, these should not be construed as limitations on the scope of the invention, but rather as an example of one embodiment thereof. Accordingly, the scope of the invention should be determined not by the embodiments illustrated, but by the appended claims and their equivalents. Table 1 : Curation of 72 metabolic genes

Recommended Uniform Screening Panel (RUSP)’

Condition NCBI NBS

ACMG code Conditions Category Gene Primary analyte type Gene method

PCCA 5095 DBS,

PROP Propionic Acidemia core Metab-OA C3

PCCB 5096 MS/MS

DBS,

MUT Methylmalonic Acidemia (mut-0, mut-) core Metab-OA MUT 4594 C3

MS/MS

MMAA 166785 DBS,

Cbl-A,B Methylmalonic Acidemia (Cobalamin disorders) core Metab-OA C3

MMAB 326625 MS/MS

DBS,

IVA Isovaleric Acidemia core Metab-OA IVD 3712 C5

MS/MS

MCCC1 56922 DBS,

3-MCC 3-Methylcrotonyl-CoA Carboxylase Deficiency core Metab-OA C5-OH

MCCC2 64087 MS/MS

DBS,

HMG 3-Hydroxy-3-Methylglutaric Aciduria core Metab-OA HMGCL 3155 C5-OH

MS/MS

HLCS 3141 DBS,

MOD Holocarboxylase Synthase deficiency core Metab-OA C5-OH

BTD 686 MS/MS

DBS,

BKT Beta-Ketothiolase Deficiency core Metab-OA ACAT1 38 C5-OH

MS/MS

DBS,

GA1 Glutaric Acidemia Type I core Metab-OA GCDH 2639 C5-DC

MS/MS

Carnitine Uptake Defect/Carnitine Transport DBS,

CUD core Metab-FAO SLC22A5 6584 CO

Defect MS/MS

Medium-chain Acyl-CoA Dehydrogenase DBS,

MCAD core Metab-FAO ACADM 34 C8

Deficiency MS/MS

Very Long-chain Acyl-CoA Dehydrogenase DBS,

VLCAD core Metab-FAO ACADVL 37 C14:1

Deficiency MS/MS

Long-chain L-3 Hydroxyacyl-CoA HADHA 3030 DBS,

LCHAD core Metab-FAO C16-OH

Dehydrogenase Deficiency MS/MS

HADHB 3032

HADHA 3030 DBS,

TFP Trifunctional Protein Deficiency core Metab-FAO C16-OH

HADHB 3032 MS/MS

DBS,

ASA Argininosuccinic Aciduria core Metab-AA ASL 435 Citrulline

MS/MS

DBS,

CIT Citrullinemia, Type I core Metab-AA ASS1 445 Citrulline

MS/MS

BCKDHA 593

BCKDHB 594

DBT 1629 DBS,

MSUD Maple Syrup Urine Disease core Metab-AA Leucine, Isoleucine

DLD 1738 MS/MS

BCKDK 10295

PPM1 K 152926

CBS 875

MTHFR 4524 DBS,

HCY Homocystinuria core Metab-AA Methionine

MTR 4548 MS/MS

MTRR 4552

DBS,

PKU Classical Phenylketonuria core Metab-AA PAH 5053 Phenylalanine

MS/MS

DBS, Tyrosine,

TYR-I Tyrosinemia, Type I core Metab-AA FAH 2184

MS/MS Succinylacetone

DBS,

BIOT Biotinidase Deficiency core Metab-other BTD 686 Biotidinase

enzyme

Other-

CF Cystic Fibrosis core CFTR 1080 DBS, IRT IRT

disorder

DBS, Galactose, GALT

GALT Classical Galactosemia core Metab-other GALT 2592

enzyme activity

MMACHC 25974 DBS,

Cbl-C,D Methylmalonic acidemia with homocystinuria secondary Metab-OA C3

MMADHC 27249 MS/MS

DBS,

MAL Malonic acidemia secondary Metab-OA MLYCD 23417 C3-DC

MS/MS

DBS,

IBG/IBD Isobutyrylglycinuria secondary Metab-OA ACAD8 27034 C4

MS/MS

DBS,

2MBG 2-Methylbutyrylglycinuria secondary Metab-OA ACADSB 36 C5

MS/MS

AUH 549

DBS,

3MGA 3-Methylglutaconic aciduria secondary Metab-OA OPA3 80207 C50H

MS/MS

TAZ 131 1 18

DBS,

2M3HBA 2-Methyl-3-hydroxybutyric aciduria secondary Metab-OA HSD17B10 3028 C50H

MS/MS

DBS,

SCAD Short-chain acyl-CoA dehydrogenase deficiency secondary Metab-FAO ACADS 35 C4

MS/MS

Medium/short-chain L-3-hydroxyacyl-CoA DBS,

M/SCHAD secondary Metab-FAO HADH 3033 C40H

dehydrogenase deficiency MS/MS

ETFA 2108

DBS,

GA-II Glutaric acidemia type II secondary Metab-FOA ETFB 2109 C4

MS/MS

ETFDH 21 10

HADHA 3030 DBS,

MCAT Medium-chain ketoacyl-CoA thiolase deficiency secondary Metab-FAO C160H

HADHB 3032 MS/MS

DBS,

CPT-IA Carnitine palmitoyltransferase type I deficiency secondary Metab-FAO CPT1A 1374 C0/C16+18

MS/MS

DBS,

CPT-II Carnitine palmitoyltransferase type II deficiency secondary Metab-FAO CPT2 1376 C16

MS/MS

DBS,

CACT Carnitine acylcarnitine translocase deficiency secondary Metab-FAO SLC25A20 788 C16

MS/MS

DBS,

ARG Argininemia secondary Metab-AA ARG1 383 Arginine

MS/MS

DBS,

CIT-II Citrullinemia, type II secondary Metab-AA SLC25A13 10165 Citrulline

MS/MS

MAT1 A 4143

DBS,

MET Hypermethioninemia secondary Metab-AA AHCY 191 Methionine

MS/MS

GNMT 27232

DBS,

H-PHE Benign hyperphenylalaninemia secondary Metab-AA PAH 5053 Phenylalanine

MS/MS

GCH1 2643 _DBS

BIOPT(BS) Biopterin defect in cofactor biosynthesis secondary Metab-AA Phenylalanine

PTS 5805 MS/MS

QDPR 5860 DBS,

BIOPT(REG) Biopterin defect in cofactor regeneration secondary Metab-AA Phenylalanine

PCBD1 5092 MS/MS

DBS,

TYR-II Tyrosinemia, type II secondary Metab-AA TAT 6898 Tyrosine

MS/MS

DBS,

TYR-III Tyrosinemia, type III secondary Metab-AA HPD 3242 Tyrosine

MS/MS

Other- DBS,

GALE Galactoepimerase deficiency secondary GALE 2582 Galactose

disorder galactose

Other- DBS,

GALK Galactokinase deficiency secondary GALK1 2584 Galactose

disorder galactose

Additional RUSPseq conditions (Not on the RUSP)*

DBS, Citrulline. Screened

(OTC) Ornithine transcarbamylase deficiency Metab-AA OTC 5009

MS/MS in CA since 2010.

No NBS. Included (CPS) Carbamoyl-phosphate synthetase deficiency - Metab-AA CPS1 1373 due to

Hyperammonemia.

No NBS. Included

(NAGSD) N-acetylglutamate synthase deficiency - Metab-AA NAGS 162417 due to

Hyperammonemia.

Methylmalonic aciduria and homocystinuria, Cbl- DBS, Related to Cbl-A,B

(Cbl-F) Metab-OA LMBRD1 55788

F type MS/MS and Cbl-C,D.

DBS, No NBS. Included

(CMAMMA) Combined malonic and methylmalonic aciduria Metab-OA ACSF3 197322

MS/MS due to MMA.

DBS, No NBS. Included (MCEE) Methylmalonyl-CoA epimerase deficiency Metab-OA MCEE 84693

MS/MS due to MMA.

AMT 275 No NBS. Included

(NKH) Nonketotic hyperglycemia Metab-AA due to DLD (PDH

GLDC 2731 complex).

Reference: www.hrsa.gov/advisory-committees/heritable-disorders/rusp/in dex.html

Table 2: Target and Primer SEQ ID NOs

Table 4: DNA Sequencing and Metabolic Data Analysis in 80 Newborns

DBS ID Conditio RUSPseq P/LP in 8 M MA genes VUS in 8 MMA PMID for 8 P/LP in 64 PMID for 64 n _ genes MMA RUSPseq genes

MUT (c.C682T:p.R228X) ^# 15643616,

(c.C581T:p.P194L); LMBRD1 20631720,

A1 mut° MUT: P/LP

(c.G1464A:p.W488X); MMACHC 27060300,

(c. A452G : p . H 151 R) ^# 27233228

MUT (c.T 1620A:p.C540X) ^# ,

19862841 ,

MUT: P/LP, MMAA:

B1 (c.C322T:p.R108C) ^# ; M MAA

mut° 16281286,

P/P (c.G3A:p.M1 l), 20301409

(c.931 delA:p.K311fs)

15643616,

C1 mut° MUT: P/P MUT (c.C982T:p.L328F*Hom) ^# -

25125334

MUT 15781192,

(c.678_679insAATTTATG:p.V227 23045948,

D1 mut° MUT: P/LP fs) ^# , (c. G607A: p. G203R) ^# ; 10923046,

LMBRD1 (c.G879A:p.W293X); 19375370,

MMAB (c.137delC:p.P46fs) 17432548

MUT

E1 mut° MUT: LP/LP (c. C 1843A : p. P615T) , (c. C422T : p. - 28973083

A141V); MCEE (c.-245-1 G>A)

MUT (c.1846C>T

F1 mut° MUT:P/VUS MUT (c. C682T : p. R228X) ^# 15643616

:p.R616C)

G1 mut° QC: low read depth

27167370,

25736335,

MUT (c.C693G:p.Y231X) ^# , 23430940,

H1 mut° MUT: P/P

C.13990T (p.R467X) ^# 15643616,

12402345,

22727635

MUT (c.G607A:p.G203R*Hom) ^# ; 10923046,

A2 mut° MUT: P/P MMAB (c.T2A:p.M1 K); MMADHC - 19375370,

(c.509delA: p. N 170fs) 17432548

_mut0 False negative (MUT: ^ _{(c C454T; p R152X)#}

B2 ClinVar

PAH

MUT (c.1891delG:p.A631fs) ^# , 16281286, (c.G1243A:p.D

9781015,

C2 mut° MUT: P/P (c.678_679insAATTTATG:p.V227 15781192, 415N) ^# ,

7556322 fs) ^# ; MMAB (c.135-2A>G) 23045948 (c.C498A:p.Y1

66X)

MUT

(c.2194_2197delGCCG/insAAGG 15643616,

D2 mut° MUT: P/P

T) ^# , (c. C682T : p. R228X) ^# ; MMAA 10923046

(c.A829G:p.R277G)

20301409,

MUT (c.C322T:p.R108C) ^# ,

E2 mut° MUT: LP/P 16281286,

(c.C682T:p.R228X) ^# 15643616

MUT (c.G607A:p.G203R*Hom) ^# ; 10923046,

F2 mut° MUT: LP/LP MMACHC 19375370,

(c.401_402del:p.D134fs) 17432548

MUT (c.G1560C:p.K520N*Hom) ^# ;

G2 mut° MUT: P/P 17075691

MMAB (c.T426A:p.Y142X)

MMACHC

Cbl

H2 MMACHC: P/LP (c.326_329del : p. P 109fs) ^# , ClinVar

(c.G482A:p.R161Q) ^#

19370762,

Cbl MMACHC (c.271 dupA: p. V90fs) ^# , 20631720,

A3 MMACHC: P/P

C,D,F (c.C615G:p.Y205X) ^# 25687216,

20631720

Cbl MMACHC (c.82-2A>G), 19370762,

B3 MMACHC: P/P

C,D,F (c.G609A:p.W203X) ^# 1631 1595

MMACHC (c.C28T:p.Q10X),

(c.T578C:p.L193P) ^# ,

Cbl 19370762,

C3 MMACHC: P/LP/P

C,D,F (c.G608A:p.W203X) ^# ; MCEE 1631 1595

(c.G428A:p.R143H); MUT

(c.G1897A:p.V633l)

co CO CO CO CO N N N N N

Q XI CD X < GO O Q XI G4 Control

H4 Control

A5 Control

B5 Control MMADHC (c.T610C:p.F204L)

C5 Control

D5 Control

E5 Control

F5 Control

G5 Control MMAA (c.562+2T>C)

H5 Control MUT (c.C890T:p.T297l)

A6 Control MMAB (c.305delT:p.L102X)

B6 Control

C6 Control

D6 Control

E6 Control MMACHC (c.259-2A>T)

F6 Control

G6 Control

PAH

(c.A204T:p.R6

26666653,

H6 Control 8S) ^# 23764561 ,

(c.A286T:p.K9

23500595 6X) (variants in

cis)

A7 Control MMAA (c.C1114T:p.Q372X)

B7 Control

A8 MMA.FP MMAA (c.G1079C:p.R360P)

MLYCD

(c.C886T:p.Q2

B8 MMA.FP MMAB (c.644+1G>C) 96X), (c.799- -

2A>T)

(variants in cis)

C8 MMA.FP

D10 MMA.FP

FAH

LMBRD1 (c.788delT:p.V

LMBRD1 : P/VUS LMBRD1 (c.C1321T:p.Q441X);

E10 MMA.FP (c.C1242T:p.C414 263fs),

(variants in cis) MMAB (c.621 delG:p.A207fs);

C) (c.930delG:p.Q

310fs)

LMBRD1 (c.T2C:p.M1T);

F10 MMA.FP

MMACHC (c.362delC:p.A121fs)

CPS1

(c.G741A:p.W

G10 MMA.FP LMBRD1 (c.G1464A:p.W488X) - 247X),

(c.C1891T:p.Q 631 X)

MUT

MUT: P/LP (variants in

H10 MMA.FP (c.1818del A : p . K606fs) , (c. G 1810A - cis)

:p.V604l)

DBT

(c.A31 G:p.S1 1

A11 MMA.FP - G),

(c.889delA:p.l2

97fs)

B11 MMA.FP

QC: failed DNA

C1 1 MMA.FP

extraction

GALT

D11 MMA.FP MMAA (c.534_535del:p.A178fs) (c.569delG:p.

W190fs)

NAGS(c.732de

IC:p.D244fs),

(c.1399_1400i

E11 MMA.FP

nsC:p.D467fs),

(c.A1605G:p.X

535W)

F11 MMA.FP -

Condition: mut° = methylmalonyl-CoA mutase mut 0 enzymatic subtype; Cbl C,D,F = cobalamin deficieny type C, D and F. Control=healthy controls sample. FP=false positive case

RUSPseq: Reportable finding with gene name and identified variants. P=pathogenic; LP=Likely pathogenic; VUS=variant of unknown significance; QC=quality control.

P/LP in 8 MMA genes: Gene names in bold indicate genes identified with two P/LP in 1 gene. (#)=known pathogenic variant in ClinVar or PubMed.

VUS in 8 MMA genes: VUS are reported for TP and FP samples with only 1 P/LP in a MMA gene.

P/LP in 64 RUSPseq genes: Variants identified in 64 genes other than the 8 MMA genes. (#)=known pathogenic variant in ClinVar or PubMed.