Quantitative amplicon sequencing for multiple copy number variation detection and allelic ratio quantification

Abstract

Provided herein is a quantitative amplicon sequencing method, in which each strand of targeted genomic loci in a DNA sample is labeled with an oligonucleotide barcode sequence by polymerase chain reaction to amplify genomic regions for high-throughput sequencing.This method can be used for simultaneous detection of copy number variations (CNV) in a series of genes of interest by quantifying the frequency of excess copies of each gene.Furthermore, these methods provide quantification of the allele ratios of different genetic identities for targeted genomic loci using multiplex PCR.Furthermore, these methods provide detection of mutations and quantification of mutant allele frequencies.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 788,375, filed January 4, 2019, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. R01 HG008752 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] Sequence Listing Reference This application contains a Sequence Listing, which is provided in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on November 26, 2019, is named RICEP0058WO_ST25.txt and is 145.6 kilobytes in size.

[0004] 1. Field The present invention relates generally to the fields of molecular biology and medicine, and more particularly to compositions and methods for multiplexed copy number variation detection and allele assignment quantification using quantitative amplicon sequencing. [Background technology]

[0005] 2. Description of Related Art Copy number variations (CNVs) are important cancer biomarkers involved in cancer formation and progression. They are present in a significant proportion of tumors, ranging from 3% to 98% depending on the cancer type. Many CNVs confer sensitivity or resistance to targeted therapies, e.g., MET amplification confers increased sensitivity to MET TKIs in non-small cell lung cancer, and PTEN deletion confers BRAF inhibitor resistance in melanoma. In tumor samples, CNVs of specific genes may only be present in a small percentage of cells (<10%) due to tumor heterogeneity and normal cell contamination.

[0006] Unlike mutations and indels, CNVs are not unique sequences, and therefore their detection requires accurate quantification, which is made difficult by chance in the sampling of DNA molecules. For example, the standard deviation (σ) of 1200 molecules per locus (i.e., 1200 haploid genome copies from 600 normal cells, 4 ng of genomic DNA) is a Poisson distribution:

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[0007] The current standard method for CNV detection in molecular diagnostics is in situ hybridization (ISH), which can determine CNV status based on the observation of a small number of cells. However, ISH technology lacks the ability to perform simultaneous analysis of a large number of genomic regions, due to the limited number of distinguishable hues in both fluorescent and bright-field microscopy. Furthermore, ISH is a complex process that needs to be performed by specialized laboratories, preventing it from being widely adopted.

[0008] Another method for CNV detection is droplet digital PCR (ddPCR), which is a PCR-based method for absolute quantification of DNA molecules. However, its limit of detection (LoD) for CNV is about 20% overcopy with many replicates. Similar to ISH, ddPCR also suffers from the inability to multiplex due to the limited number of fluorescent channels. Microarray-based methods, including array comparative genomic hybridization and SNP arrays, are highly multiplexed methods used for screening many CNVs and aneuploidies. However, they are not good at detecting small CNVs of <40kb or low-frequency CNVs of <30% overcopy.

[0009] Next-generation sequencing (NGS) is a high-throughput technology that has shown rapid cost declines over the past decade. NGS is common in the field of cancer molecular diagnostics. Highly multiplexed mutation detection with LoD of <0.1% variant allele frequency has been achieved and commercialized with NGS platforms. However, the current LoD of NGS methods for CNV detection is not excellent, and whole-exome sequencing (WES), which has been used for CNV discovery at a level of about 30% overcopy, is expensive and requires even more NGS reads (with a proportional increase in cost) to achieve a lower LoD. Smaller hybrid-capture panels, such as the FoundationOne commercial panel, can achieve a LoD of about 30% overcopy at a lower cost.

[0010] In diagnostic NGS panels, target enrichment is necessary to reduce NGS reads wasted on unrelated genomic regions. Two common methods for target enrichment are hybrid-capture and multiplex PCR. Most current NGS-based CNV panels are hybrid-capture based, meaning that the target regions are captured by biotinylated nucleic acid probes and separated from the rest of the genome using streptavidin magnetic beads. Hybrid-capture panels have low predictive value when the panel size is small, so most panels are >100kb (i.e., >1000 probes or loci), which is due to non-specific binding of unwanted DNA on the bead surface, probes, and captured targets. Due to the large number of loci, the coverage of hybrid-capture panels is not uniform, with the 95% and 5% percentile loci differing by at least 30-fold, introducing another layer of bias into the quantification. Hybrid-capture panels also suffer from low conversion rates (i.e., the proportion of input molecules sequenced) caused by imperfect end-repair and ligation, a biased sampling process, and contribute to variability. Summary of the Invention

[0011] Provided herein is a method of quantitative amplicon sequencing, in which each strand of targeted genomic loci in a DNA sample is labeled with an oligonucleotide barcode sequence by polymerase chain reaction to amplify genomic regions for high-throughput sequencing.The method can be used for simultaneous detection of copy number variation (CNV) in a set of genes of interest by quantifying the frequency of overcopy of each gene.Furthermore, these methods provide quantification of the allele ratio of different genetic identities for targeted genomic loci using multiplex PCR.

[0012] In one embodiment, provided herein is a method for preparing a targeted region of genomic DNA for high throughput sequencing, the method including: (a) obtaining a genomic DNA sample; and (b) (i) sequencing a first region, 0 to 50 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, a second region having a length of 0 to 50 nucleotides (e.g., 1, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides), a third region having at least four degenerate nucleotides (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 degenerate nucleotides), and a fourth region comprising a sequence that is complementary to the first target genomic DNA region; and (ii) from 5' to 3', a fifth region, 0 to 50 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). (c) amplifying at least a portion of the genomic DNA sample by performing two cycles of PCR using a second oligonucleotide comprising a sixth region having a length of at least 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides) and a seventh region comprising a sequence complementary to the second target genomic DNA region; at an annealing temperature 0-10°C (e.g., 1-10, 2-10, 3-10, 4-10, 5-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-9, 2-8, 2-7°C, or any range or value that can be derivable therein) higher than the annealing temperature used, and using (i) a third oligonucleotide comprising a sequence capable of hybridizing to a reverse complement of at least a portion of the first region, and (ii) a fourth oligonucleotide comprising a sequence capable of hybridizing to a reverse complement of at least a portion of the fifth region,amplifying the product of step (b) by performing at least three cycles of PCR; and (d) amplifying, from 5' to 3', the eighth region, 0 to 50 nucleotides (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 108, 109 and amplifying the product of step (c) by performing at least one cycle of PCR using a fifth oligonucleotide comprising a ninth region having a length of at least 10 nucleotides (45, 46, 47, 48, 49, or 50 nucleotides) and a tenth region comprising a sequence complementary to the third target genomic DNA region, wherein the third target genomic DNA region is at least one nucleotide closer to the first target genomic DNA than the second target genomic DNA region.

[0013] In some embodiments, the method is a method for preparing 1 to 10,000 targeted regions of genomic DNA for high throughput sequencing (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 250, 500, 750, 1,000, 2,000, 3,000, 4,000, or 5,000, and up to 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 750, 500, 250, 100, 75, or 50 targeted regions, or any range or value that can be derived therein). In some embodiments, the third region is a unique molecular identifier (UMI). In some embodiments, the third target genomic DNA region is 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) bases closer to the first target genomic DNA region than the second target genomic DNA region. In some embodiments, the first region and the eighth region are universal primer binding sites. In some embodiments, the first region and the eighth region are full or partial NGS adaptor sequences. In some embodiments, the fifth region comprises a sequence that cannot be found in the human genome. In some embodiments, the fifth region comprises a sequence that is different from the NGS adaptor sequence. In some embodiments, the melting temperatures of the first and fifth regions are 0-10° C. (e.g., 1-10, 2-10, 3-10, 4-10, 5-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2-9, 2-8, 2-7° C., or any range or value derivable therein) higher than the melting temperatures of the fourth and seventh regions. In some embodiments, the degenerate nucleotides in the third region are each independently one of A, T, or C. In some embodiments, no degenerate nucleotides in the third region are G. In some embodiments, there is a population of first oligonucleotides each having a unique third region.

[0014] In some embodiments, the method further comprises purifying the product of step (c). In some embodiments, the purifying comprises SPRI purification or column purification. In some embodiments, the method further comprises purifying the product of step (d). In some embodiments, the purifying comprises SPRI purification or column purification. In some embodiments, the method further comprises (e) amplifying the product of step (d) by PCR using primers that hybridize to the first region and the eighth region, the primers comprising index sequences for next generation sequencing. In some embodiments, the method further comprises purifying the product of step (e). In some embodiments, the purifying comprises SPRI purification or column purification. In some embodiments, the method further comprises (f) performing high throughput DNA sequencing of the product of step (e). In some embodiments, the high throughput DNA sequencing comprises next generation sequencing.

[0015] In some embodiments, the first target genomic DNA region and the second target genomic DNA region are on opposite strands of genomic DNA. In some embodiments, the first target genomic DNA region and the second target genomic DNA region are separated by 40 nucleotides to 500 nucleotides (e.g., 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides, or any range and value derived therein). In some embodiments, step (b) comprises an extension time of about 30 minutes (e.g., 27, 28, 29, 30, 31, 32, or 33 minutes). In some embodiments, step (c) comprises an extension time of about 30 seconds (e.g., 27, 28, 29, 30, 31, 32, or 33 seconds). In some embodiments, step (d) comprises an extension time of about 30 minutes (e.g., 27, 28, 29, 30, 31, 32, or 33 minutes).

[0016] In some embodiments, provided herein is a method for quantifying the frequency of overcopy (FEC) of at least one target gene, the method comprising: (a) obtaining a genomic DNA sample; (b) preparing the genomic DNA for high-throughput sequencing according to any one of the methods of the present embodiments, wherein sequences of the fourth region, the seventh region, and the tenth region hybridize to the at least one target gene; (c) performing high-throughput sequencing according to any one of the methods of the present embodiments; and (d) calculating the FEC for the at least one target gene based on the sequencing information obtained in step (c).

[0017] In some embodiments, the method is for quantifying FEC for a set of target genes, the set of target genes including between 2 and 1000 target genes (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 250, 500, or 750, and up to 1,000, 900, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 targeted regions, or any ranges and values ​​derived therein). In some embodiments, step (b) is carried out using a first oligonucleotide group, a second oligonucleotide group, and a fifth oligonucleotide group, and a portion of each of the first, second, and fifth oligonucleotide groups comprises a fourth, seventh, and tenth region that is complementary to one of a set of target genes, respectively. In some embodiments, each of the fourth, seventh, and tenth regions comprises a sequence that is found only once in the human genome. In some embodiments, each first oligonucleotide that hybridizes to one target gene has a unique third region compared to each other first oligonucleotide that hybridizes to the same target gene. In some embodiments, step (b) is carried out using a first oligonucleotide, a second oligonucleotide, and a fifth oligonucleotide that comprises a fourth, seventh, and tenth region that is complementary to a reference gene, respectively. In some embodiments, step (b) prepares a portion of each target gene or reference gene for high-throughput sequencing, the portion being between 40 nucleotides and 500 nucleotides in length (e.g., 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides, or any range and value derivable therein).

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[0018] In some embodiments, step (d) includes: (i) aligning NGS reads with the targeted portion of each target gene, and grouping NGS reads into subgroups based on the loci to which they align; (ii) classifying NGS reads at each locus based on their UMI sequence, such that all NGS reads carrying the same UMI sequence are grouped as one UMI family; (iii) removing UMI families resulting from PCR errors or NGS errors; (iv) counting the number of unique UMI sequences at each locus; and (v) calculating FEC based on the number of unique UMIs at each locus in each target gene and reference gene. In some embodiments, step (d)(iii) includes removing UMI sequences that do not fit the UMI degenerate base design. In some embodiments, step (d)(iii) comprises removing UMI families having a UMI family size smaller than Fmin, where UMI family size is the number of reads carrying the same UMI, and Fmin is between 2 and 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, step (d)(iv) comprises removing UMI sequences that differ by only 1 or 2 bases from another UMI sequence having a larger family size.

[0019] In some embodiments, the FEC is:

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[0020] In one embodiment, provided herein is a method for quantifying allele ratios of different genetic identities for at least one target genomic locus, the method comprising: (a) obtaining a genomic DNA sample; (b) preparing the genomic DNA for high-throughput sequencing according to any one of the methods of the present embodiments, wherein sequences of the fourth region, the seventh region, and the tenth region hybridize to the genomic DNA near at least one target gene; (c) performing high-throughput sequencing according to any one of the methods of the present embodiments; and (d) calculating the allele ratios of different genetic identities for the at least one target genomic locus based on the sequencing information obtained in step (c).

[0021] In some embodiments, the method is for identifying allelic ratios of different genetic identities for a set of target genomic loci, where the set of target genomic loci includes between 2 and 10,000 target genomic loci (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 250, 500, 750, 1,000, 2,000, 3,000, 4,000, or 5,000, and up to 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, 750, 500, 250, 100, 75, or 50 target genomic loci, or any range or value derivable therein). In some embodiments, step (b) is carried out using a first group of oligonucleotides, a second group of oligonucleotides, and a fifth group of oligonucleotides, and each of the first, second, and fifth groups of oligonucleotides comprises a fourth, seventh, and tenth region that is complementary to genomic DNA near at least one of a series of target genomic loci, respectively. In some embodiments, each of the fourth, seventh, and tenth regions comprises a sequence that cannot hybridize to non-target regions of genomic DNA under the conditions of step (b). In some embodiments, each first oligonucleotide that hybridizes to genomic DNA near one target genomic locus has a unique third region compared to each other first oligonucleotide that hybridizes to genomic DNA near the same target genomic locus. In some embodiments, each target genomic locus is between 40 nucleotides and 500 nucleotides in length (e.g., 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 nucleotides, or any ranges and values ​​derivable therein).

[0022] In some embodiments, step (d) includes: (i) aligning the NGS reads with the targeted genomic loci and grouping the NGS reads into subgroups based on the loci to which they align; (ii) classifying the NGS reads at each locus based on their UMI sequence, such that all NGS reads carrying the same UMI sequence are grouped as one UMI family; (iii) removing UMI families resulting from PCR or NGS errors; (iv) determining the genetic identity for each remaining UMI family; (v) counting the number of unique UMI sequences at each locus; and (vi) calculating the allele ratio. In some embodiments, step (d)(iii) includes removing UMI sequences that do not fit the UMI degenerate base design. In some embodiments, step (d)(iii) comprises removing UMI families with a UMI family size smaller than Fmin, where UMI family size is the number of reads carrying the same UMI, and Fmin is 2 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, step (d)(iii) comprises removing UMI sequences that differ from another UMI sequence with a larger family size by only 1 or 2 bases. In some embodiments, step (d)(iv) comprises determining genetic identity only if at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, or 98%) of the reads in the UMI family are the same at the genetic locus of interest. In some embodiments, the allelic ratio is calculated based on the R 対立遺伝子 =N1 / N2, where N1 is the number of unique UMIs for the first genetic identity and N2 is the number of unique UMIs for the second genetic identity.

[0023] In some embodiments, step (d)(iv) comprises identifying a consensus sequence for each UMI family. In some embodiments, the consensus sequence is the sequence that occurs most frequently in the UMI family. In some embodiments, the method further comprises comparing the consensus sequence to the wild-type sequence for the locus, thereby identifying a mutation in the consensus sequence. In some embodiments, the method further comprises calculating the variant allele frequency (VAF) of the identified mutation. In some embodiments, the VAF of the identified mutation is defined as the number of UMI families with the mutation / total number of UMI families.

[0024] As used herein, "essentially free" with respect to a named component is used herein to mean that none of the named components are intentionally incorporated into the composition and / or are present as contaminants or in only trace amounts. Thus, the total amount of the named components resulting from unintentional contamination of a composition is well below 0.05%, preferably below 0.01%. Most preferred are compositions in which the amount of a specific component cannot be analyzed using standard analytical methods.

[0025] As used herein, "a" or "an" may mean one or more. When used in the claims, when used in conjunction with the term "including," the terms "a" or "an" may mean one or more than one.

[0026] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" may mean at least a second or more.

[0027] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, method employed to determine the value, or the variation that exists among testing subjects.

[0028] [The present invention 1001] 1. A method for preparing a targeted region of genomic DNA for high throughput sequencing, comprising: (a) obtaining a genomic DNA sample; (b)(i) a first oligonucleotide comprising, from 5' to 3', a first region, a second region having a length of 0 to 50 nucleotides, a third region comprising at least four degenerate nucleotides, and a fourth region comprising a sequence complementary to a first target genomic DNA region; and (ii) a second oligonucleotide comprising, from 5' to 3', a fifth region, a sixth region having a length of 0 to 50 nucleotides, and a seventh region comprising a sequence complementary to a second target genomic DNA region; amplifying at least a portion of the genomic DNA sample by performing two cycles of PCR using (c) an annealing temperature that is 0 to 10° C. higher than the annealing temperature used in step (b); and (i) a third oligonucleotide comprising a sequence capable of hybridizing to a reverse complement of at least a portion of the first region; and (ii) a fourth oligonucleotide comprising a sequence capable of hybridizing to a reverse complement of at least a portion of the fifth region; amplifying the product of step (b) by performing at least three cycles of PCR using (d) a fifth oligonucleotide comprising, from 5' to 3', an eighth region, a ninth region having a length of 0 to 50 nucleotides, and a tenth region comprising a sequence complementary to a third target genomic DNA region; amplifying the product of step (c) by performing at least one cycle of PCR using wherein the third target genomic DNA region is at least one nucleotide closer to the first target genomic DNA region than the second target genomic DNA region. [The present invention 1002] 1001. The method of the present invention, which is a method for preparing 1 to 10,000 targeted regions of genomic DNA for high-throughput sequencing. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein the third region is a unique molecular identifier (UMI). [The present invention 1004] The method according to any one of claims 1001 to 1003, wherein the third target genomic DNA region is closer to the first target genomic DNA region by 1 to 10 bases than the second target genomic DNA region. [The present invention 1005] The method according to any one of claims 1001 to 1004, wherein the first region and the eighth region are universal primer binding sites. [The present invention 1006] Any of the methods of 1001 to 1005, wherein the first region and the eighth region comprise a complete or partial NGS adapter sequence. [The present invention 1007] The method of any one of claims 1001 to 1006, wherein said fifth region comprises a sequence that cannot be found in the human genome. [The present invention 1008] The method of any one of claims 1001 to 1007, wherein the fifth region comprises a sequence different from the NGS adaptor sequence. [The present invention 1009] The method according to any one of claims 1001 to 1008, wherein the melting temperatures of the first region and the fifth region are 0 to 10°C higher than the melting temperatures of the fourth region and the seventh region. [The present invention 1010] The method of any one of claims 1001 to 1009, wherein said degenerate nucleotides in said third region are each independently one of A, T, or C. [The present invention 1011] The method of any of claims 1001 to 1010, wherein none of the degenerate nucleotides in the third region is G. [The present invention 1012] The method of any of claims 1001 to 1011, wherein there is a population of first oligonucleotides, each having a unique third region. [The present invention 1013] The method of any one of claims 1001 to 1012, further comprising purifying the product of step (c). [The present invention 1014] The method of the present invention 1013, wherein the purifying comprises SPRI purification or column purification. [The present invention 1015] The method of any one of claims 1001 to 1014, further comprising purifying the product of step (d). [The present invention 1016] The method of claim 1015, wherein the purifying comprises SPRI purification or column purification. [The present invention 1017] (e) amplifying the product of step (d) by PCR using primers that hybridize to the first region and the eighth region, the primers comprising index sequences for next-generation sequencing. Any of the methods of 1001 to 1016 of the present invention, further comprising: [The present invention 1018] The process of claim 1017, further comprising purifying the product of step (e). [The present invention 1019] The method of claim 1018, wherein the purifying comprises SPRI purification or column purification. [The present invention 1020] (f) performing high throughput DNA sequencing of the product of step (e). Any of the methods of 1017 to 1019 of the present invention further comprises: [The present invention 1021] High throughput DNA sequencing includes next generation sequencing. [The present invention 1022] The method according to any one of claims 1001 to 1021, wherein said first target genomic DNA region and said second target genomic DNA region are on opposite strands of said genomic DNA. [The present invention 1023] The method according to any one of claims 1001 to 1022, wherein the first target genomic DNA region and the second target genomic DNA region are separated by 40 nucleotides to 500 nucleotides. [The present invention 1024] The method of any one of claims 1001 to 1023, wherein step (b) comprises an extension time of about 30 minutes. [The present invention 1025] The method of any one of claims 1001 to 1024, wherein step (c) comprises an extension time of about 30 seconds. [The present invention 1026] The method of any one of claims 1001 to 1025, wherein step (d) comprises an extension time of about 30 minutes. [The present invention 1027] 1. A method for quantifying the frequency of overcopy (FEC) of at least one target gene, comprising: (a) obtaining a genomic DNA sample; (b) preparing the genomic DNA for high-throughput sequencing according to any one of the methods of the present invention 1001 to 1026, wherein the sequences of the fourth region, the seventh region, and the tenth region hybridize to the at least one target gene; (c) performing high-throughput sequencing according to the method of the present invention 1020; (d) calculating the FEC for the at least one target gene based on the sequence information obtained in step (c); The method comprising: [The present invention 1028] The method of claim 1027, wherein the method is for quantifying the FEC for a set of target genes, and the set of target genes comprises 2 to 1000 target genes. [The present invention 1029] The method of any one of claims 1027 to 1028, wherein step (b) is carried out using a first population of oligonucleotides, a second population of oligonucleotides, and a fifth population of oligonucleotides, each of which includes a fourth, seventh, and tenth region, respectively, that is complementary to one of the set of target genes. [The present invention 1030] 1029. The method of any of claims 1027 to 1029, wherein each of said fourth, seventh, and tenth regions comprises a sequence found only once in the human genome. [The present invention 1031] Any of the methods of claims 1027 to 1030, wherein each first oligonucleotide that hybridizes to one target gene has a unique third region compared to each other first oligonucleotide that hybridizes to the same target gene. [The present invention 1032] Any of the methods of claims 1027 to 1031, wherein step (b) is carried out using a first oligonucleotide, a second oligonucleotide, and a fifth oligonucleotide, each of which comprises a fourth, seventh, and tenth region that is complementary to the reference gene. [The present invention 1033] 1032. The method of any of claims 1027 to 1032, wherein step (b) comprises preparing a portion of each target or reference gene for high-throughput sequencing, said portion being between 40 nucleotides and 500 nucleotides in length. [The present invention 1034] The FEC: TIFF0007602464000007.tif11128 Any of the methods of claims 1027 to 1033 of the present invention, defined as [The present invention 1035] Step (d) is (i) aligning NGS reads to the targeted portion of each target gene and grouping the NGS reads into subgroups based on the locus to which they align; (ii) classifying the NGS reads at each locus based on their UMI sequence such that all NGS reads carrying the same UMI sequence are grouped into one UMI family; (iii) removing UMI families resulting from PCR or NGS errors; and (iv) counting the number of unique UMI sequences at each locus; and (v) calculating the FEC for each locus in each target gene and reference gene based on the number of unique UMI sequences; Any of the methods of 1027 to 1034 of the present invention. [The present invention 1036] The method of the present invention, wherein step (d)(iii) comprises removing UMI sequences that do not fit the UMI degenerate base design. [The present invention 1037] The method of any one of claims 1035 to 1036, wherein step (d)(iii) comprises removing UMI families having a UMI family size smaller than Fmin, the UMI family size being the number of reads carrying the same UMI, and Fmin being between 2 and 20. [The present invention 1038] Any of the methods of 1035 to 1037, wherein step (d)(iv) comprises removing UMI sequences that differ from another UMI sequence having a larger family size by only one or two bases. [The present invention 1039] The FEC: TIFF0007602464000008.tif11128 is defined as: TIFF0007602464000009.tif6128 is the sum of the number of unique UMIs for all or a portion of the target loci, u is the number of loci under consideration, u is less than or equal to the total number of loci in the target locus, TIFF0007602464000010.tif7128 Any of the methods of claims 1027 to 1038, wherein v is the sum of the number of unique UMIs for all or a portion of the reference loci, v is the number of loci considered for one reference, v is less than or equal to the total number of loci in said reference, w is the number of references considered, w is less than or equal to the total number of references, and k is determined by experimental calibration. [The present invention 1040] Any of the methods of claims 1027 to 1039, wherein the FEC is used to identify a copy number variation (CNV) status of the target gene. [The present invention 1041] 1. A method for quantifying allelic ratios of different genetic identities for at least one target genomic locus, comprising: (a) obtaining a genomic DNA sample; (b) preparing the genomic DNA for high-throughput sequencing according to any one of the methods of the present invention 1001 to 1026, wherein the sequences of the fourth region, the seventh region, and the tenth region hybridize to the genomic DNA near the at least one target genomic locus; (c) performing high-throughput sequencing according to the method of the present invention 1020; (d) calculating allele ratios of different genetic identities for the at least one target genomic locus based on the sequencing information obtained in step (c); The method comprising: [The present invention 1042] The method of the present invention, wherein the method is for quantifying the allelic ratios of different genetic identities for a set of target genomic loci, and the set of target genomic loci comprises 2 to 10,000 target genomic loci. [The present invention 1043] The method of claim 1041 or 1042, wherein step (b) is carried out using a first population of oligonucleotides, a second population of oligonucleotides, and a fifth population of oligonucleotides, each of which includes a fourth, seventh, and tenth region, respectively, that is complementary to the genomic DNA near at least one of the set of target genomic loci. [The present invention 1044] Any of the methods of claims 1041 to 1043, wherein each of the fourth, seventh, and tenth regions comprises a sequence that cannot hybridize to a non-target region of the genomic DNA under the conditions of step (b). [The present invention 1045] Any of the methods of claims 1041 to 1044, wherein each first oligonucleotide that hybridizes to the genomic DNA near a target genomic locus has a unique third region compared to each other first oligonucleotide that hybridizes to the genomic DNA near the same target genomic locus. [The present invention 1046] 1046. The method of any one of claims 1041 to 1045, wherein each target genomic locus is between 40 nucleotides and 500 nucleotides in length. [The present invention 1047] Step (d) is (i) aligning NGS reads to the targeted genomic loci and grouping the NGS reads into subgroups based on the loci to which they align; (ii) classifying the NGS reads at each locus based on their UMI sequence such that all NGS reads carrying the same UMI sequence are grouped into one UMI family; (iii) removing UMI families resulting from PCR or NGS errors; and (iv) determining said genetic identity for each remaining UMI family; and (v) counting the number of unique UMI sequences at each locus; and (vi) calculating said allelic ratio; Any of the methods of 1041 to 1046 of the present invention. [The present invention 1048] The method of claim 10, wherein step (d)(iii) comprises removing UMI sequences that do not fit the UMI degenerate base design. [The present invention 1049] The method of any one of claims 1047 to 1048, wherein step (d)(iii) comprises removing UMI families having a UMI family size smaller than Fmin, said UMI family size being the number of reads carrying the same UMI, and Fmin being between 2 and 20. [The present invention 1050] Any of the methods of 1047 to 1049, wherein step (d)(iii) comprises removing UMI sequences that differ from another UMI sequence having a larger family size by only one or two bases. [The present invention 1051] Any of the methods of claims 1047 to 1050, wherein step (d)(iv) comprises determining the genetic identity only if at least 70% of the reads in a UMI family are identical at the genetic locus of interest. [The present invention 1052] The allelic ratio is R 対立遺伝子 =N 1 / N 2 where N 1 is the number of unique UMIs for the first genetic identity, and N 2 Any of the methods of claims 1041 to 1051, wherein x is the number of unique UMIs for the second genetic identity. [The present invention 1053] Any of the methods of claims 1047 to 1051, wherein step (d)(iv) comprises identifying a consensus sequence for each UMI family. [The present invention 1054] The method of claim 10, wherein said consensus sequence is the sequence that occurs most frequently in said UMI family. [The present invention 1055] The method of any one of claims 1053 to 1054, further comprising comparing said consensus sequence to a wild-type sequence for said locus, thereby identifying mutations in said consensus sequence. [The present invention 1056] The method of any one of claims 10 to 55, further comprising calculating the variant allele frequency (VAF) of said identified mutations. [The present invention 1057] The method of claim 1056, wherein the VAF of the identified mutation is defined as the number of UMI families carrying the mutation / total number of UMI families. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given for illustrative purposes only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0029] The accompanying drawings form part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0030] (Figure 1) Schematic of QASeq primer design and experimental workflow. Each primer set contains three different oligos: specific forward primer (SfP), specific reverse primer A (SrPA), and specific reverse primer B (SrPB). Each QASeq panel only requires one universal forward primer (UfP) and one universal reverse primer (UrP). There may be additional bases at the 5' end of region 1 or region 5 in UfP or UrP. In one recommended workflow, the DNA sample is first mixed with all of SfP, SrPA, DNA polymerase, dNTPs, and PCR buffer. Two cycles of long extension PCR are performed for the addition of UMIs at all target loci. The annealing temperature is then increased by approximately 8°C above the PCR amplification temperature for approximately 7 cycles using UfP and UrP (short extension, approximately 30 s) to amplify the molecules while preventing the addition of multiple UMIs to the same original molecule; note that the addition of UfP and UrP to the reaction is an open-tube step in the thermocycler. After purification using SPRI magnetic beads or columns, SrPB primers, DNA polymerase, dNTPs, and PCR buffer are mixed with the PCR products for adapter replacement, and after two cycles of long extension (approximately 30 min), NGS adapters are added only to the correct PCR products, not primer-dimers or non-specific products. After another purification using SPRI magnetic beads or columns, a standard NGS index PCR is performed to normalize and load the libraries onto an Illumina sequencer. (Figure 2) Simulation of UMI cross-bonding energy. UMI as (N) 20 Or (SWW)6SW instead of (H) 20 Using the method, sequences lower the average cross-linking energy and exhibit little primer-dimer interaction. Here, 500 simulations were performed for each UMI pattern, and in each simulation, two sequences matching the pattern were randomly generated, and the cross-linking ΔG° between these sequences was reduced by 60°C and 0.18MK. + The calculation was made assuming the following. (Figure 3A-B) Spacers between primers and UMIs reduce PCR bias. (Figure 3A) Workflow to evaluate the importance of spacers between primers and UMIs. Input molecules were amplified separately using three sets of primers: no spacer (set 1), a 5 nt spacer between forward primer and UMI and a 5 nt spacer between reverse primer and UMI (set 2), or a 12 nt spacer between forward primer and UMI and an 11 nt spacer between reverse primer and UMI (set 3). Indexing was performed prior to NGS analysis by Illumina MiSeq. (Figure 3B) Experimental UMI family size distribution histograms for the three sets of primers. UMI sequences that did not match the UMI design pattern were removed. (Figure 4A-B) Data analysis for UMI-based absolute quantification of CNVs. (Figure 4A) Data analysis workflow for CNV detection. NGS reads in the FASTQ output file are analyzed to obtain the CNV status as a result. The FEC of the target gene is

number

number

number

[0031] Detailed Description Provided herein is a method of quantitative amplicon sequencing, in which each strand of targeted genomic loci in original DNA sample is labeled with oligonucleotide barcode sequence by polymerase chain reaction to amplify genomic regions for high-throughput sequencing.Also provided herein is a method that allows simultaneous detection of copy number variation (CNV) in a series of genes of interest by quantifying the frequency of excess copies of each gene.The method of the present disclosure also provides quantification of the allele ratio of different genetic identities for targeted genomic loci using multiplex PCR.These methods can be applied to the detection of CNV in genes of interest in tumor samples, which can guide the selection of targeting therapy and help understand cancer formation and progression.

[0032] The current standard method for prenatal diagnosis of monogenic diseases is to sequence fetal genetic material obtained from invasive and risky chorionic villus bioscopy or amniocentesis. Noninvasive prenatal genetic testing (NIPT) for monogenic diseases is based on circulating fetal-derived cell-free DNA (cfDNA) in maternal plasma. The presence of background maternal DNA makes it difficult to confidently detect allele ratio changes arising from fetal cfDNA, especially when the maternal DNA is heterozygous at the locus of interest. Droplet digital PCR (ddPCR) has been used to quantify the allele ratio between mutant alleles carrying disease-causing mutations and wild-type alleles in NIPT (Lun et al., 2008), but the practical feasibility is limited by the accuracy and reliability of the technique. QASeq allows absolute quantification of DNA molecules by appending a unique molecular identifier to each strand of the original input molecule, and can be applied to allele ratio quantification in NIPT. Therefore, QASeq can also be used for allele ratio quantification. Allele ratio quantification aims to quantify the ratio of DNA molecules with different genetic identities. Accurate allele ratio quantification is the key to NIPT of monogenic diseases such as β-thalassemia and cystic fibrosis.

[0033] I. Frequency of CNV excess copies The frequency of extra copies (FEC) of CNVs in genomic DNA samples was as follows:

number

[0034] Although QASeq can be used to quantify FEC, it does not provide information about the percentage of cells containing CNVs in a tumor tissue sample. For example, if 1% of cells in a tumor sample contain 4 copies of ERBB2 and the remaining 99% of cells contain 2 copies, the FEC is 1%, and if 0.5% of cells in a tumor sample contain 6 copies of ERBB2 and the remaining 99.5% of cells contain 2 copies, the FEC is still 1%. Furthermore, QASeq does not provide information about the genomic location of the extra copies.

[0035] II. Multiplex PCR Panel Design In a QASeq multiplex PCR panel, one target gene requires M (M = 1-1000) sets of primers, each amplifying a non-overlapping small region (40nt-500nt, usually ≦200nt) in the target gene region. When a panel has multiple target genes, the number of primer sets used in each gene is similar (approximately M). The panel also contains a similar number of primer sets (approximately M) amplifying a reference genomic region. The reference loci serve as an internal standard in the amount of genomic DNA (gDNA) loaded, thereby not requiring precise quantification of DNA concentration in the sample. At least one reference primer set may be used in each panel. Increasing the number of input molecules or loci in the target gene can both reduce mutations in random sampling, so a larger number of primer sets per gene can be used to improve the LoD for sample types containing smaller amounts of DNA, and the number of reference primer sets needs to be increased proportionally in this case.

[0036] Each primer set contains three different oligos: a specific forward primer (SfP), a specific reverse primer A (SrPA), and a specific reverse primer B (SrPB) (see Figure 1). SfP contains regions 1, 2, 3, and 4 from 5' to 3'. Region 4 is the template binding region, region 3 is the UMI region, region 1 is a full or partial NGS adapter, and region 2 is an optional spacer region (typically 0-15 nt) added for uniform amplification of the UMI. SrPA contains regions 5, 6, and 7 from 5' to 3'. Region 7 is the template binding region, region 5 is a custom adapter for universal amplification (i.e., a sequence not found in the human genome, unlike NGS adapters), and region 6 is an optional spacer region (typically 0-15 nt) added for uniform amplification of different loci. SrPB contains regions 8, 9, and 10 from 5' to 3'. Region 10 is the template binding region, whose 3' end is at least one base closer to region 4 than region 7, region 8 is a full or partial NGS adaptor, and region 9 is an optional spacer region (typically 0-15 nt) added for uniform amplification of different loci. Each QASeq panel requires only one universal forward primer (UfP) and one universal reverse primer (UrP). UfP includes region 1, UrP includes region 5, and there may be additional bases at the 5' end of region 1 or region 5 in UfP or UrP. The melting temperatures (Tm) of template binding regions 4, 7, and 10 are approximately the same as the PCR annealing temperature, and the Tm of UfP and UrP is not lower than that of regions 4, 7, and 10 in the experimental PCR conditions.

[0037] When designing primers, single nucleotide polymorphisms (SNPs) with significantly less allele frequency (MAF) should be avoided in the primer binding region, so that the binding affinity of the primers will not be affected by nucleotide sequence variations in different patient samples. Furthermore, the entire human genome nucleotide sequence should be searched to ensure that the primers are not prone to non-specific amplification of non-target regions.

[0038] In an exemplary panel targeting ERBB2 CNVs in formalin-fixed paraffin-embedded (FFPE) specimens of tumor samples, 10 sets of primers, each amplifying a 60-70 nt amplicon, were designed in the ERBB2 gene region. In addition, 10 sets of reference primers were designed, each amplifying a region in a different housekeeping gene from a different chromosome (Table 1). Primers were automatically designed using Matlab code to minimize primer interactions while fulfilling the above design principles. Additionally, non-pathogenic SNPs with >0.2% MAF in the population were avoided. An online tool, Primer-BLAST, was used to ensure that each primer set had only one amplicon in the human genome. Primer sequences are shown in Table 2.

[0039] (Table 1) Amplicon location TIFF0007602464000015.tif58150

[0040] Table 2: Primer sequences in exemplary QASeq panels TIFF0007602464000016.tif122150TIFF0007602464000017.tif223150TIFF0007602464000018.tif224150TIFF0007602464000019.tif47150

[0041] Table 3 Primer sequences for 179-plex wide-range plates TIFF0007602464000020.tif162150TIFF0007602464000021.tif230150TIFF0007602464000022.tif230150TIFF0007602464000023.tif230150TIFF0007602464000024.tif230150TIFF0007602464000025.tif230150TIFF0007602464000026.tif230150TIFF0007602464000027.tif227150TIFF0007602464000028.tif230150TIFF0007602464000029.tif228150TIFF0007602464000030.tif228150TIFF0007602464000031.tif228150TIFF0007602464000032.tif228150TIFF0007602464000033.tif228150TIFF0007602464000034.tif227150TIFF0007602464000035.tif230150TIFF0007602464000036.tif230150TIFF0007602464000037.tif230150TIFF0007602464000038.tif230150TIFF0007602464000039.tif230150TIFF0007602464000040.tif230150TIFF0007602464000041.tif230150TIFF0007602464000042.tif139150

[0042] III. UMI Design In the NGS library preparation process, the PCR amplification step can significantly increase quantification variation, making it difficult to identify small changes in the number of original molecules. Using UMI technology, PCR bias can be reduced to achieve absolute quantification of original DNA molecules. The concept of UMI is to give every original DNA molecule a different DNA sequence as a "barcode", so that the origin of each NGS read can be traced based on the barcode sequence. When enough NGS reads are obtained, the number of unique UMIs found in the NGS output can reflect the number of original DNA molecules. Previously, UMI technology was mainly used for error correction in NGS-based detection of low frequency mutations. It has also been applied to quantification. Uniquely labeling each original molecule is achieved by using a large number of different UMI sequences, e.g., 10 for 100,000 original molecules. 9 Using 20 different UMI sequences results in <0.006% of molecules carrying repeating UMIs.

[0043] DNA sequences containing degenerate bases such as poly(N) (i.e., a mix of A, T, C, or G at each position) are often used as UMI sequences. In QASeq, poly(H) (A, T, or C) is used as UMI because it has a weaker cross-linking energy compared to poly(N) or a mix of S (C or G) and W (A or T) bases, as shown by simulations (Figure 2). (H) 20 is 3.5 x 10 9 (H) contains different sequences, is sufficient for 100,000 molecules as input, 15 is 1.4 × 10 7 It contains different sequences and is sufficient for 6,000 molecules as input.

[0044] IV. Spacers to reduce PCR bias PCR efficiency varies for amplicons with different sequences. Because the UMI consists of many different sequences, a spacer between the primer and the variable UMI region can be used to achieve more uniform PCR efficiency.

[0045] NGS was performed to evaluate the effect of spacers on PCR bias (Figure 3A). The template molecule had two adapters at the 5' and 3' ends for amplification, and the UMI region was located in the middle (D). 15 The templates were amplified separately using three sets of primers: no spacer (set 1), a 5 nt spacer between the forward primer and the UMI and a 5 nt spacer between the reverse primer and the UMI (set 2), or a 12 nt spacer between the forward primer and the UMI and an 11 nt spacer between the reverse primer and the UMI (set 3). Indexes were added via PCR prior to NGS analysis. (D) 15 is 1.4 × 10 7 Each NGS read contains distinct sequences. Because the number of input template molecules is much smaller than the number of possible sequences, each unique UMI sequence only has one copy before amplification. All NGS reads carrying the same UMI are likely derived from the same molecule. Therefore, the UMI family size (i.e., the number of reads carrying the same UMI) is an indicator of PCR efficiency.

[0046] UMI family size distributions were compared to assess the significance of spacers in PCR bias (Figure 3B). The longer the spacer between the primer and the UMI, the more uniform the distribution was observed. In primer set 3, where the spacer length was longer than 10 nt on both ends, a significantly improved distribution was achieved.

[0047] V. QASeq Workflow The outline of the QASeq NGS library preparation workflow is shown in Figure 1. First, the DNA sample is mixed with SfP, SrPA, DNA polymerase, dNTPs, and PCR buffer. Two cycles of long extension (~30 min) PCR are performed for UMI addition at all target loci. After that, each strand in one DNA molecule will carry a different UMI. Next, to amplify the molecule while preventing the addition of multiple UMIs to the same original molecule, the annealing temperature is increased by 8°C and amplification is performed with UfP and UrP for at least two cycles (e.g., ~7 cycles) with short extension (~30 s). The addition of UfP and UrP to the reaction is a tube opening step in the thermocycler. After purification using SPRI magnetic beads or columns, mix SrPB primer, DNA polymerase, dNTPs, and PCR buffer with the PCR products for adapter replacement, and after at least one cycle (e.g., two cycles) of long extension (~30 min), NGS adapters are added only to the correct PCR products, not primer-dimers or non-specific products. After another purification using SPRI magnetic beads or columns, perform a standard NGS index PCR to normalize and load the libraries onto an Illumina sequencer.

[0048] All types of DNA polymerases and PCR supermixes can be used. Standard annealing, extension, and denaturation temperatures for the specific polymerase used should be followed (except for universal PCR, which increases the annealing temperature).

[0049] VI. Alternative QASeq Workflows The workflow could be performed using SfP and SrPB to add UMIs using two cycles of PCR, and then directly adding the index primer for index PCR. To test this, 20 sets of SfP and SrPB were used in the same reaction. The experimental hit rate of the method was very low (0.5%), so the method may not be useful for diagnostic NGS assays (Figure 9A). Off-target NGS reads were mostly primer dimers. In a second alternative workflow, universal PCR was performed using UfP and Urp for 6 cycles of universal PCR, followed by a purification step. These additional steps improved the hit rate to 12-28% (average hit rate = 20%) for the different libraries (Figure 9B). A third alternative workflow based on the second alternative workflow was tested. In this, a size selection step using agarose gel was added after index PCR to further reduce primer dimers. The experimental average hit rate improved to 42%, but was still lower than 50% (Figure 9C). Primer dimer reduction was achieved using the first experimental workflow, including both adapter replacement and purification after universal PCR, resulting in a high average hit rate of 66% (Figure 9D). One source of primer dimers in the above workflow is shown in Figure 9E. If the 3' portion of SfP binds to SfPB, or the 3' portion of SfPB binds to SfP, a dimer strand with universal regions at both the 5' and 3' ends can be generated and therefore amplified in the universal or index PCR step.

[0050] The first workflow includes a final index step that adds an index sequence and a sequencer P5 / P7 sequence to the end of the amplicon, but there are alternative workflows that add the sequences during UMI addition, universal PCR, or adapter replacement steps, and thus do not require an index PCR step. Figures 10A-C show three examples. First, an index and P5 sequence are added to the 5' of UfP, and another index and P7 sequence are added to the 5' of SrPB. The amplicon resulting from adapter replacement contains P5, P7, and a double index, and is therefore ready for sequencing (Figure 10A). Second, an index and P7 sequence are added to the 5' of SrPB, and this modified SrPB is mixed with a normal P5 index primer in the adapter replacement step (Figure 10B). Third, an index and P5 sequence are added to the 5' of SfP, and a primer carrying the P5 sequence is used as UfP in the universal PCR step. Another index and P7 sequence are added 5' to SrPB (Figure 10C).

[0051] The alternative QASeq primer design and workflow is shown in Figure 11. Each primer set contains three different oligos: a specific forward primer (SfP), a specific reverse primer A (SrPA), and a specific reverse primer B (SrPB). SfP contains regions 1, 2, 3, and 4 from 5' to 3'. Region 4 is the template binding region, region 3 is the UMI region, region 1 is a full or partial NGS adapter, and region 2 is an optional spacer region (0-15 nt) that is added for uniform amplification of the UMI. SrPA contains region 5, which is the template binding region. SrPB contains regions 6, 7, and 8 from 5' to 3'. Region 8 is the template binding region, whose 3' end is at least one base closer to region 4 than region 5; region 6 is a full or partial NGS adaptor; and region 7 is an optional spacer region (0-15 nt) added for uniform amplification of different loci. Each QASeq panel only requires one universal forward primer (UfP), including region 1, and there may be an additional base at the 5' end of region 1 in UfP. The melting temperature (Tm) of template binding regions 4, 5, and 8 is approximately the same as the PCR annealing temperature, and the Tm of UfP is not lower than regions 4, 5, and 8 under experimental PCR conditions. Compared to the original design, only SrPA needs the template binding region, and the universal reverse primer (UrP) is not required. In the experimental workflow, more cycles of PCR (e.g., at least 10 cycles) are required for the universal PCR step under this alternative primer design.

[0052] VII. Data Analysis Workflow A schematic of the data analysis workflow for CNV detection is shown in Figure 4A. First, raw NGS data are aligned to amplicon regions, and optional adapter trimming can be performed before alignment. Non-aligned reads are discarded, and aligned reads are grouped by the loci to which they align.

[0053] Then, all reads aligned to the same locus are further assigned by UMI sequence, i.e., reads carrying the same UMI are grouped as one UMI family. The UMI family size is the number of reads carrying the same UMI, and the number of unique UMIs is the total number of different UMI sequences at one locus (Figure 4B). Then, all unique UMI families that may be the result of PCR or NGS errors are removed. For example, UMI sequences that do not match the designed UMI pattern (e.g., G bases found in poly(H)UMI sequences) are erroneous and should be removed. Furthermore, if two UMI sequences differ by only 1-2 bases, the one with the smaller UMI family size may have been mutated from the other and therefore may be optionally removed. After removal of UMI errors, the family size is <F min The UMI family with F min is determined based on the distribution of UMI family sizes, and F min = 4 may be the most common example used. The number of unique UMIs (N) after UMI removal is used in the next step.

[0054] The FEC of the target gene is:

number

number

number

[0055] VIII. Allelic Ratio Quantification QASeq can be applied to quantify allelic ratios of different genetic identities for 1-10,000 genomic loci using multiplex PCR. The multiplex PCR panel design for targeted genomic loci and the experimental workflow for labeling each strand of the targeted genomic loci with oligonucleotide barcode sequences by PCR, followed by amplification of the genomic region for high-throughput sequencing, are similar to CNV detection.

[0056] An outline of the data analysis workflow for allelic ratio quantification is shown in Figure 12A. First, raw NGS data are aligned to amplicon regions, and optional adapter trimming can be performed before alignment. Non-aligned reads are discarded, and aligned reads are grouped by the loci to which they align. At each locus, NGS reads are assigned by UMI, and all NGS reads carrying the same UMI sequence are grouped as one UMI family. Unique UMI families with errors in UMIs may be the result of PCR or NGS errors, and are removed as described in the data analysis workflow section.

[0057] Genetic identity (wild type or mutation) in each remaining UMI family is determined based on majority vote, and genetic identity must be supported by at least 70% of the members (reads) in the same UMI family. As an example in Figure 12B, in a UMI family with UMI family size = 7, all 7 reads share the same UMI sequence (indicated by 2D barcode). Genetic identity at the locus of interest is "A" in 6 reads and "G" in 1 read. Since more than 70% of the reads in the UMI family support "A", the genetic identity in this UMI family is called "A". The 1 read corresponding to "G" is the result of a PCR or NGS error. UMIs that do not have more than 70% of the reads supporting one common genetic identity are discarded.

[0058] Then, the number of unique UMIs, N (the total number of different UMI sequences at one locus), is counted for each different genetic identity at the targeted locus, where N denotes the number of original strands. The allelic ratio at the targeted locus is R 対立遺伝子 =N1 / N2, where N1 is the number of unique UMIs for the first genetic identity and N2 is the number of unique UMIs for the second genetic identity.

[0059] IX.Definitions "Amplification," as used herein, refers to any in vitro process for increasing the number of copies of a nucleotide sequence or sequences. Nucleic acid amplification results in the incorporation of nucleotides into DNA or RNA. As used herein, one amplification reaction can consist of many rounds of DNA replication. For example, one PCR reaction can consist of 30-100 "cycles" of denaturation and replication.

[0060] "Polymerase chain reaction", or "PCR", refers to a reaction for in vitro amplification of a specific DNA sequence by simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for multiple copies or replication of a target nucleic acid flanked by primer binding sites, which includes one or more repetitions of (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers in the presence of nucleoside triphosphates by a nucleic acid polymerase. Usually, the reaction is cycled in a thermal cycler device with different temperatures optimized for each step. The specific temperatures, durations at each step, and variation rates between steps depend on many factors well known to those skilled in the art, and are exemplified, for example, by McPherson et al., editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively).

[0061] "Primer" refers to an oligonucleotide, either natural or synthetic, that, when it forms a duplex with a polynucleotide template, can act as an initiation point for nucleic acid synthesis and can be extended from its 3' end along the template, thereby forming an extended duplex. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Typically, the primer is extended by a DNA polymerase. Primers are generally of a length compatible with their use in synthesizing primer extension products, and typically range from 8-100 nucleotides in length, e.g., 10-75, 15-60, 15-40, 18-30, 20-40, 21-50, 22-45, 25-40, etc., and more commonly range from 18-40, 20-35, 21-30 nucleotides in length, and any length between the recited ranges. Exemplary primers can range anywhere from 10 to 50 nucleotides in length, such as 15 to 45, 18 to 40, 20 to 30, 21 to 25, and any length between the recited ranges. In some embodiments, primers typically do not exceed about 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

[0062] As used herein, "incorporating" means becoming a part of a nucleic acid polymer.

[0063] The term "in the absence of exogenous manipulation" as used herein refers to the modification of nucleic acid molecules occurring without changing the solution in which the nucleic acid molecules are modified. In certain embodiments, it occurs without the presence of human hands or machines that change the solution conditions, which may also be referred to as buffer conditions. However, changes in temperature may occur during modification.

[0064] A "nucleoside" is a base-sugar combination, i.e., a nucleotide that lacks a phosphate. Certain interchangeability in the use of the terms nucleoside and nucleotide is recognized in the art. For example, the nucleotide deoxyuridine triphosphate, dUTP, is a deoxyribonucleoside triphosphate. After incorporation into DNA, it functions as a DNA monomer and is formally deoxyuridylic acid, i.e., dUMP or deoxyuridine monophosphate. When dUTP is incorporated into DNA, it can be said that the resulting DNA does not have a dUTP portion. Similarly, when deoxyuridine is incorporated into DNA, it can be said that it is only a part of the substrate molecule.

[0065] As used herein, "nucleotide" refers to a base-sugar-phosphate combination. Nucleotides are monomeric units of nucleic acid polymers, i.e., DNA and RNA. This term includes ribonucleotide triphosphates such as rATP, rCTP, rGTP, or rUTP, and deoxyribonucleotide triphosphates such as dATP, dCTP, dUTP, dGTP, or dTTP.

[0066] The term "nucleic acid" or "polynucleotide" generally refers to at least one molecule or strand of DNA, RNA, DNA-RNA chimera, or derivatives or analogs thereof, including at least one nucleic acid base, such as a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A", guanine "G", thymine "T", and cytosine "C") or RNA (e.g., A, G, uracil "U", and C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide". As used herein, "oligonucleotide" refers collectively and interchangeably to two terms in the art, "oligonucleotide" and "polynucleotide". It is noted that although oligonucleotide and polynucleotide are different terms in the art, there is no precise dividing line between them, and they are used interchangeably herein. The term "adapter" may also be used interchangeably with the terms "oligonucleotide" and "polynucleotide". In addition, the term "adapter" may refer to a linear adapter (either single-stranded or double-stranded) or a stem-loop adapter. These definitions generally refer to at least one single-stranded molecule, but in certain embodiments also encompass at least one additional strand that is partially, substantially, or completely complementary to at least one single-stranded molecule. Thus, a nucleic acid can encompass at least one double-stranded molecule or at least one triplex molecule that includes one or more complementary strands or "complements" of a particular sequence comprising a strand of the molecule. As used herein, single-stranded nucleic acids can be represented by the prefix "ss", double-stranded nucleic acids by the prefix "ds", and triple-stranded nucleic acids by the prefix "ts".

[0067] "Nucleic acid molecule" or "nucleic acid target molecule" refers to any single-stranded or double-stranded nucleic acid molecule that contains standard basic bases, per-modified bases, unnatural bases, or any combination of those bases. For example, and without limitation, a nucleic acid molecule contains the four standard DNA bases-adenine, cytosine, guanine, and thymine, and / or the four standard RNA bases-adenine, cytosine, guanine, and uracil. Uracil can be substituted for thymine when the nucleoside contains a 2'-deoxyribose group. A nucleic acid molecule can be converted from RNA to DNA and from DNA to RNA. For example, and without limitation, mRNA can be generated into complementary DNA (cDNA) using reverse transcriptase, and DNA can be generated into RNA using RNA polymerase. A nucleic acid molecule can be of biological or synthetic origin. Examples of nucleic acid molecules include genomic DNA, cDNA, RNA, DNA / RNA hybrids, amplified DNA, existing nucleic acid libraries, and the like. Nucleic acids can be obtained from human samples, including blood, serum, plasma, cerebrospinal fluid, cheek scrape, biopsy, semen, urine, feces, saliva, sweat, etc. Nucleic acid molecules can be subjected to various treatments, such as repair and fragmentation treatments. Fragmentation treatments include mechanical, sonic, and hydrodynamic shearing. Repair treatments include nick repair via extension and / or ligation, blunting to produce blunt ends, removal of damaged bases, such as deamination, derivatization, abasic, or cross-linked nucleotides, etc. Nucleic acid molecules of interest can also be subjected to chemical modification (e.g., bisulfite conversion, methylation / demethylation), extension, amplification (e.g., PCR, isothermal, etc.), etc.

[0068] A "complementary" or "complementary" nucleic acid is one that can base pair according to standard Watson-Crick, Hoogsteen or non-Hoogsteen binding complementarity rules. As used herein, the term "complementary" or "complementary" can refer to a nucleic acid that is substantially complementary when it can be evaluated by the same nucleotide comparison as above. The term "substantially complementary" refers to a nucleic acid that includes at least one sequence of consecutive nucleic acid bases, or semi-consecutive nucleic acid bases when one or more nucleic acid base portions are not present in the molecule, and can hybridize to at least one nucleic acid strand or double strand, even if less than all of the nucleic acid bases do not base pair with the corresponding nucleic acid base. In certain embodiments, a "substantially complementary" nucleic acid comprises at least one sequence in which about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, and any ranges thereof, of the nucleic acid sequence can form base pairs with at least one single-stranded or double-stranded nucleic acid during hybridization. In certain embodiments, the term "substantially complementary" refers to at least one nucleic acid that can hybridize under stringent conditions with at least one nucleic acid strand or double strand. In certain embodiments, a "partially complementary" nucleic acid comprises at least one sequence that can hybridize under less stringent conditions with at least one single-stranded or double-stranded nucleic acid, or comprises at least one sequence in which less than about 70% of the nucleobase sequence can base pair with at least one single-stranded or double-stranded nucleic acid molecule during hybridization.

[0069] The term "non-complementary" refers to a nucleic acid sequence that lacks the ability to form at least one Watson-Crick base pair through specific hydrogen bonding.

[0070] The term "degenerate" as used herein refers to a nucleotide or a series of nucleotides whose identity can be selected from a variety of nucleotides as the opposite of a given sequence. In certain embodiments, there can be a selection from two or more different nucleosides. In further specific embodiments, the selection of nucleotides at one particular position includes a selection from only purines, only pyrimidines, or non-pairing purines and pyrimidines.

[0071] "Sample" refers to a material obtained or isolated from a fresh or preserved biological sample or synthetically produced source that contains the nucleic acid of interest. Samples include at least one cell, fetal cell, cell culture, tissue specimen, blood, serum, plasma, saliva, urine, tears, vaginal secretions, sweat, lymphatic fluid, cerebrospinal fluid, mucosal secretions, peritoneal fluid, ascites, feces, body exudates, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, multicellular embryo, lysate, extract, solution, or reaction mixture suspected of containing the immune nucleic acid of interest. Samples can also include non-human sources, such as non-human primates, rodents, other mammals, other animals, plants, fungi, bacteria, and viruses.

[0072] As used herein in reference to a nucleotide sequence, "substantially known" refers to having sufficient sequence information to allow for the preparation of a nucleic acid molecule, including amplification. This is typically about 100%, but in some embodiments, some portion of the adaptor sequence is random or degenerate. Thus, in certain embodiments, substantially known refers to about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.

[0073] X. Further Processing of Target Nucleic Acid A. DNA Amplification Many template-dependent processes are available for amplifying the nucleic acid present in a given template sample.One of the most well-known amplification methods is the polymerase chain reaction (also called PCR™), which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159, and Innis et al., 1990, each of which is incorporated herein by reference in its entirety.Briefly, two synthetic oligonucleotide primers that are complementary to two regions of template DNA (one for each strand) are added to template DNA (not necessarily pure) in the presence of excess deoxynucleotides (dNTPs) and a thermostable polymerase, such as Taq (Thermus aquaticus) DNA polymerase. In a series of temperature cycles (typically 30-35), the target DNA is repeatedly denatured (at about 90°C) and annealed (generally at 50-60°C) to primers and daughter strands extended from the primers (72°C). As daughter strands are generated, they act as templates in subsequent cycles. Thus, the template region between the two primers amplifies exponentially rather than linearly.

[0074] B. DNA Sequencing Methods are also provided for sequencing the library of adaptor-ligated fragments.Any technique for sequencing nucleic acids known to those skilled in the art can be used in the disclosed method.DNA sequencing techniques include classical dideoxy sequencing reaction (Sanger method) using labeled terminators or primers and gel separation in slabs or capillaries, sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, allele-specific hybridization with a library of labeled oligonucleotide probes, sequencing by synthesis using allele-specific hybridization with a library of labeled clones followed by ligation, real-time monitoring of the incorporation of labeled nucleotides during the polymerization step, and SOLiD sequencing.

[0075] The nucleic acid library can be created by a method compatible with Illumina sequencing, such as the Nextera™ DNA Sample Preparation Kit, and additional methods for creating Illumina next-generation sequencing library preparations are described, for example, in Oyola et al. (2012). In other embodiments, the nucleic acid library is created by a method compatible with SOLiD™ or Ion Torrent sequencing (e.g., SOLiD™ Fragment Library Construction Kit, SOLiD™ Mate-Paired Library Construction Kit, SOLiD™ ChIP-Seq Kit, SOLiD™ Total RNA-Seq Kit, SOLiD™ SAGE™ Kit, Ambion™ RNA-Seq Library Construction Kit, etc.). Additional methods for next-generation sequencing include various methods for library construction that can be used in embodiments of the present invention, and are described, for example, in Pareek (2011) and Thudi (2012).

[0076] In certain embodiments, the sequencing technology used in the method of the present disclosure includes HiSeq™ system (e.g., HiSeq™ 2000 and HiSeq™ 1000), NextSeq™ 500, and MiSeq™ system of Illumina, Inc. The HiSeq™ system is based on the attachment of randomly fragmented genomic DNA to a planar optically transparent surface and the massively parallel sequencing of millions of fragments using solid-phase amplification to create a high-density sequencing flow cell with millions of clusters, each containing about 1,000 copies of template per square centimeter. These templates are sequenced using four-color DNA sequencing by synthesis technology. The MiSeq™ system uses TruSeq™, Illumina's reversible terminator-based sequencing by synthesis.

[0077] Another example of a DNA sequencing technique that can be used in the disclosed method is 454 sequencing (Roche) (Margulies et al., 2005). 454 sequencing involves two steps. In the first step, DNA is sheared into fragments of about 300-800 base pairs, and the fragments are blunt-ended. Oligonucleotide adapters are then ligated to the ends of the fragments. The adapters serve as primers for amplification and sequencing of the fragments. The fragments can be attached to DNA capture beads, e.g., streptavidin-coated beads, using, e.g., adapter B, that contains a 5'-biotin tag. The bead-bound fragments are PCR amplified within droplets of an oil-water emulsion. The result is multiple copies of clonally amplified DNA fragments on each bead. In the second step, the beads are captured in wells (picoliter size). Pyrosequencing is performed on each DNA fragment in parallel. The addition of one or more nucleotides results in a light signal that is recorded by a CCD camera in the sequencing device. The signal intensity is proportional to the number of nucleotides incorporated.

[0078] Another example of a DNA sequencing technology that can be used in the disclosed method is the SOLiD technology (Life Technologies, Inc.). In the SOLiD sequencing technology, genomic DNA is sheared into fragments and adapters are attached to the 5' and 3' ends of the fragments to generate a fragment library. Alternatively, internal adapters can be introduced by ligating adapters to the 5' and 3' ends of the fragments, circularizing the fragments, digesting the circularized fragments to generate internal adapters, and attaching adapters to the 5' and 3' ends of the resulting fragments to generate a paired library. A clonal bead population is then prepared in a microreactor containing beads, primers, templates, and PCR components. After PCR, the templates are denatured to enrich the beads and separate beads with extended templates. The templates on the selected beads are subjected to a 3' modification that allows for attachment to a glass slide.

[0079] Another example of a DNA sequencing technology that may be used in the disclosed method is the Ion Torrent system (Life Technologies, Inc.). Ion Torrent uses a high-density array of micromachined wells to perform this biochemical process in a massively parallel fashion. Each well holds a different DNA template. Under the well is an ion-sensitive layer, and under that is the proprietary Ion sensor. When a nucleotide, such as C, is added to the DNA template and then incorporated into a strand of DNA, a hydrogen ion is released. The charge from that ion changes the pH of the solution and can be detected by the proprietary ion sensor. The sequencer searches for a base, going directly from chemical information to digital information. The Ion Personal Genome Machine (PGM™) sequencer sequentially fills the chip with one nucleotide after another. If the next nucleotide that fills the chip does not match, no current change is recorded and the base is not searched for. If there are two identical bases in the DNA strand, the voltage doubles and the chip records the two identical bases that were searched for. This is direct detection - no scanning, no cameras, no light - and each nucleotide incorporation is recorded in seconds.

[0080] Another example of a sequencing technology that may be used in the disclosed method includes Pacific Biosciences' Single Molecule, Real Time (SMRT™) technology. In SMRT™, each of the four DNA bases is attached to one of four different fluorescent dyes. These dyes are phospholinked. A single DNA polymerase is immobilized by one molecule of template single-stranded DNA at the bottom of a zero-mode waveguide (ZMW). The ZMW is a containment structure that allows observation of the incorporation of a single nucleotide by the DNA polymerase against a background of fluorescent nucleotides that diffuse rapidly (microseconds) into and out of the ZMW. It takes a few microseconds to incorporate the nucleotide into the growing strand. During this time, the fluorescent label is excited, producing a fluorescent signal, and the fluorescent tag is cleaved. Detection of the fluorescence of the corresponding dye indicates which base has been incorporated. The process is repeated.

[0081] Further sequencing platforms include the CGA platform (Complete Genomics). The CGA technology is based on the preparation of circular DNA libraries and rolling circle amplification (RCA), resulting in DNA nanoballs that are aligned on a solid support (Drmanacet al., 2009). Complete Genomics' CGA platform uses a novel strategy called combinatorial probe anchor ligation (cPAL) for sequencing. The process is initiated by hybridization between the anchor molecule and one of the unique adapters. Four degenerate 9-mer oligonucleotides are labeled with a specific fluorophore that corresponds to a specific nucleotide (A, C, G, or T) at the first position of the probe. Sequencing occurs in a reaction where the correctly matching probe hybridizes to the template and is ligated to the anchor using T4 DNA ligase. After imaging of the ligated product, the ligated anchor-probe molecules are denatured. The process of hybridization, ligation, imaging, and denaturation is repeated five times using a new set of fluorescently labeled 9-mer probes containing known bases at positions n+1, n+2, n+3, and n+4.

[0082] XI. Kit The technology herein includes a kit for analyzing copy number variation or allele frequency in DNA samples. "Kit" refers to a combination of physical components. For example, a kit can include one or more components, such as, for example, nucleic acid primers, enzymes, reaction buffers, instructions, and other elements that are useful for carrying out the technology described herein. These physical elements can be arranged in any manner suitable for carrying out the present invention.

[0083] The components of the kit may be packaged either in aqueous media or in lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components are placed, and preferably appropriately aliquoted (e.g., aliquoted into the wells of a microtiter plate). If there is more than one component in the kit, the kit will also generally include a second, third, or other additional container into which the additional components may be separately placed. However, various combinations of components may be included in a single vial. The kits of the present invention will also typically include a means for containing the nucleic acids, and any other reagent containers in hermetically sealed containment for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. The kits will also include instructions for using the kit components, as well as for the use of any other reagents not included in the kit. The instructions may include variations that can be implemented. EXAMPLES

[0084] XII. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the examples which follow demonstrate techniques discovered by the inventors to function well in the practice of the invention and therefore can be considered to constitute preferred modes for its practice. However, those skilled in the art should understand, in light of this disclosure, that many changes can be made in the specific embodiments disclosed which will still yield the same or similar results without departing from the spirit and scope of the invention.

[0085] Example 1 - Calibration Results An exemplary calibration experiment of the ERBB2 QASeq panel was performed on a normal cell line gDNA sample NA18562, which would not contain ERBB2 amplification, to analyze quantification variability and potential LoD. The workflow was as described in the "QASeq workflow" section. Taq polymerase was used for all PCR steps. Denaturation was performed at 95°C and annealing / extension was performed at 60°C (except for the universal PCR step, where annealing / extension was performed at 68°C). 15 reads were reserved for each molecule / UMI, since all original molecules with their bound UMIs must be present in the NGS output. For an input of 2500 haploid genome copies and a 20 amplicon panel, the total reads required is approximately 2 x 2500 x 20 x 15 = 1,500,000. Note that each strand in a DNA duplex carries a different UMI in this workflow, so 2500 haploid genome copies = 5000 molecules = 8.3 ng of gDNA. This experiment was performed on an Illumina MiSeq instrument.

[0086] NGS reads were aligned to amplicon sequences using exact strand matching, and the alignment rate was between 50% and 70% for the different libraries. Then, UMI family size and unique UMI number were analyzed. The distribution of UMI family sizes peaked at approximately 20 in the most abundant loci (Figure 5). UMI families containing obvious PCR errors (i.e., G bases found in poly(H)UMI sequences) and UMIs with family sizes <4 were removed (Figure 5). If the UMI binding rate is perfect, the number of unique UMIs should be equal to the original number of molecules in the sample. With an input of 2500 haploid genome copies (5000 molecules), a number of unique UMIs ranging from 632 to 3065 was obtained depending on the locus (Figure 6).

[0087] To estimate the LoD of this assay, libraries were prepared for four different DNA inputs: 75, 250, 750, and 2500 haploid genome copies, with each condition replicated five times. The CNV ratios of the samples were calculated as described in the "Data Analysis Workflow" section. The standard deviation of the CNV ratios over the five replicates (σ CNV比 ) to assess quantification variability, and the LoD of the assay was determined to be within 3σ CNV比 Simulations were also performed to estimate the theoretical σ CNV比 was calculated. When the number of input molecules increases, σ CNV比 Note that the LoD decreases with σ CNV比 is higher than the theoretical value (Fig. 7), which was predicted due to the inability to eliminate UMI binding bias and amplification bias. CNV比 is 1% at 2500 haploid genome copies, and conservatively, using a linear fit based on all 4 data points, σ CNV比 = 2%, and therefore the estimated LoD was approximately 6% overcopy. Based on extrapolation to 50,000 haploid genome copy input, the possible σ CNV比 The LoD was 0.3%, and the LoD was approximately 1%. Another way to assess the LoD is by testing a series of calibration samples containing different frequencies of overcopies, with the lowest detectable frequency of overcopies being the LoD.

[0088] Example 2 - CNV detection results in FFPE samples Two FFPE slides were analyzed using the exemplary ERBB2 panel described in the "Multiplex PCR Panel Design" section and in Example 1. The FFPE slides (purchased from Asterand) were obtained from the same lung cancer tumors that were not predicted to contain ERBB2 CNVs. First, DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen), yielding >6 μg DNA per sample. Libraries were prepared using the same method as described in Example 1. 8.3 ng of extracted DNA was used for each library, which corresponds to 2500 haploid genome copies and 5000 molecular input. The number of NGS reads obtained for each library (1,500,000 reads) was the same as the 2500 haploid genome copy input cell line gDNA library.

[0089] Data analysis was performed using the same method as described in Example 1. A similar pattern of UMI family size distribution was obtained as for the cell line gDNA library (Figure 8A). The number of unique UMIs was smaller than that of the cell line gDNA library with 2500 haploid genome copies input. The UMI binding yield of the FFPE samples was on average about 1 / 4 that of the cell line gDNA, indicating that more than 300% FFPE DNA needs to be loaded to achieve the same LoD as the cell line gDNA samples (Figure 8B).

[0090] The calculated CNV ratios for the FFPE samples are shown in Figure 8C. The estimated LoD of this assay = 15% is based on the calibration results with 750 haploid genome copies input cell line gDNA, which has a similar number of unique UMIs as the FFPE library. Based on the results, no CNVs of ERBB2 were detected in these FFPE slides. Since the LoD decreases with increasing number of input molecules, a LoD of 6% can be achieved based on the calibration results with 2500 haploid genome copies input cell line gDNA.

[0091] Example 3 - CNV quantification results in loaded clinical FFPE samples A 100-plex QASeq panel was used to quantify ERBB2 ploidy in breast cancer FFPE samples: 50-plex for the ERBB2 gene region (see Table 3 for primer sequences, primer names have "ERBB2" therein) and 50-plex for the short arm of chromosome 17 as a reference (see Table 3 for primer sequences, primer names have "Ref" therein).

[0092] Two previously characterized FFPE DNA samples (one "normal" and one "ERBB2 amplified abnormal") were mixed to obtain 2.5%, 5%, and 10% ERBB2 FEC samples. The "normal" sample DNA was extracted from an FFPE lung cancer sample (purchased from Asterand), which should have no ERBB2 amplification (FEC=0%), and the "ERBB2 amplified abnormal" sample DNA was extracted from an FFPE breast cancer sample (purchased from OriGene), which has an ERBB2 FEC of 78%. Sample input was 8.3ng DNA (quantified by qPCR) per library. The "normal" samples were tested separately with five replicate NGS libraries each prepared with 8.3ng DNA input. The experimentally normalized FEC values ​​are shown in FIG. 13. The normalized FEC was calculated as follows: Normalized FEC 試料 =(1+FEC 試料 ) / (1+FEC 正常試料 )-1

[0093] FEC 正常試料 was the average of five repeats. The LoD of CNVs was estimated as follows: FEC LoD =3×σ 正常試料 / (1+FEC 正常試料 )=0.85%

[0094] Here, σ 正常試料was the standard deviation of five replicates. CNVs were successfully detected in the 2.5%, 5%, and 10% ERBB2 FEC samples because their calculated FECs were outside the 3 standard deviation range (see FIG. 13). The experimentally normalized FECs of ERBB2 correlate well with the predicted values.

[0095] Example 4 - Comprehensive panel for mutation and CNV quantification The provided method (QASeq) can be used not only for CNV quantification, but also for NGS error correction and mutation quantification. In each QASeq amplicon, the region between 3' of fP and 3' of rPin is the mutation detection region (MDR), and any small mutations (including base substitutions, deletions, and insertions smaller than 500 bp) in the MDR can be detected with a LoD of 0.1%-0.3%. This is vastly superior to standard non-UMI NGS for mutation detection, which has a LoD of about 1%.

[0096] A 179-plex comprehensive panel was developed and tested for both mutation and CNV quantification in breast cancer samples. All plexes contain the three primers described in the previous section: fP (fP (aka SfP), rPin (aka SrPB), and rPout (aka SrPA). 95 primer sets were used solely for CNV quantification, including 45 sets in the gene ERBB2, and 50 sets in the short arm of chromosome 17 as a reference. Five primer sets in the ERBB2 gene were used for both CNV and mutation quantification. Another 79 primer sets were used for mutation quantification only. UfP and UrP were used for universal amplification (see Table 3 for sequences).

[0097] CNV quantification was performed in the same way as described in the previous section. The data processing workflow for mutation quantification is summarized in Figure 14. After optional adapter trimming, NGS reads were aligned with the amplicon sequences. For each locus, reads were assigned to UMI families, and UMI families with errors in the UMI sequence were removed, as well as UMI families with small UMI family size (≦3). We then found the consensus MDR sequence for each UMI family, which is usually the MDR sequence that represents the maximum number of occurrences in the UMI family. The final step was to compare the consensus sequence with the wild-type MDR sequence and perform mutation calling from scratch. The VAF of one mutation can be calculated as follows: VAF=number of UMI families with mutations / total number of UMI families.

[0098] This 179-plex panel was tested with Horizon Discovery's Multiplex I cfDNA Reference Standard Set. Triplicate NGS libraries of the Wild Type cfDNA Reference Standard and triplicate 0.3% cfDNA Reference Standard (prepared by mixing 0.1% cfDNA Reference Standard and 1% cfDNA Reference Standard) were tested. Sample input was 8.3ng DNA per library (quantified by qPCR).

[0099] Overall hit rates were greater than 50% for all libraries (i.e., >50% of NGS reads could be aligned to the amplicon), and conversion rates (i.e., percentage of input molecules sequenced) had an average of 62%, with 97% of the plexes having >10% conversion rates (see Figure 15). Error rates after UMI correction varied at different nucleotide positions, with maximum error rates of 0.23%, 0.20%, and 0.23% and average error rates of 0.006%, 0.005%, and 0.005% for libraries with Horizon Discovery Multiplex I Wild Type cfDNA Reference Standard repeated three times (see Figure 16). The mutation quantification capillary was validated using a 0.3% cfDNA Reference Standard. The experimental VAFs of the 6 mutations were generally consistent with the predicted VAFs, with the differences mostly attributable to chance in sampling a small number (≦9) of mutant molecules (see Figure 17).

[0100] All methods disclosed and claimed herein may be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made in the methods, steps, or sequence of steps described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined in the appended claims.

[0101] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are incorporated herein by reference. TIFF0007602464000046.tif17150

Claims

1. 1. A method for preparing a targeted region of genomic DNA for high throughput sequencing, comprising: (a) obtaining a genomic DNA sample; (b)(i) a first oligonucleotide comprising, from 5' to 3', a first region, a second region having a length of 0-50 nucleotides, a third region comprising at least four degenerate nucleotides, and a fourth region comprising a sequence complementary to a first target genomic DNA region, wherein the third region is a unique molecular identifier (UMI); and (ii) a second oligonucleotide comprising, from 5' to 3', a fifth region, a sixth region having a length of 0 to 50 nucleotides, and a seventh region comprising a sequence complementary to a second target genomic DNA region; amplifying at least a portion of the genomic DNA sample by performing two cycles of PCR using (c) at an annealing temperature that is 1-10° C. higher than the annealing temperature used in step (b); and (i) a third oligonucleotide comprising a sequence capable of hybridizing to a reverse complement of at least a portion of the first region; and (ii) a fourth oligonucleotide comprising a sequence capable of hybridizing to a reverse complement of at least a portion of the fifth region. amplifying the product of step (b) by performing at least three cycles of PCR using (d) a fifth oligonucleotide comprising, from 5' to 3', an eighth region, a ninth region having a length of 0 to 50 nucleotides, and a tenth region comprising a sequence complementary to the third target genomic DNA region; amplifying the product of step (c) by performing at least one cycle of PCR using wherein the third target genomic DNA region is at least one nucleotide closer to the first target genomic DNA region than the second target genomic DNA region.

2. 2. The method of claim 1, which is a method for preparing 1 to 10,000 targeted regions of genomic DNA for high throughput sequencing.

3. 3. The method of claim 1, wherein the third target genomic DNA region is 1 to 10 bases closer to the first target genomic DNA region than the second target genomic DNA region.

4. The method of any one of claims 1 to 3, wherein the first region and the eighth region are universal primer binding sites.

5. The method of any one of claims 1 to 4, wherein the first region and the eighth region comprise a complete or partial NGS adapter sequence.

6. The method of any one of claims 1 to 5, wherein the fifth region comprises a sequence that cannot be found in the human genome.

7. The method of any one of claims 1 to 6, wherein the fifth region comprises a sequence different from an NGS adapter sequence.

8. The method according to any one of claims 1 to 7, wherein the melting temperatures of the first region and the fifth region are 0 to 10°C higher than the melting temperatures of the fourth region and the seventh region.

9. 9. The method of any one of claims 1 to 8, wherein the degenerate nucleotides in the third region are each independently one of A, T, or C.

10. 10. The method of any one of claims 1 to 9, wherein none of the degenerate nucleotides in the third region is G.

11. The method of any one of claims 1 to 10, wherein there is a population of first oligonucleotides, each having a unique third region.

12. The method of any one of claims 1 to 11, further comprising purifying the product of step (c).

13. The method of claim 12, wherein the purifying comprises SPRI purification or column purification.

14. The method of any one of claims 1 to 13, further comprising purifying the product of step (d).

15. The method of claim 14 , wherein the purifying comprises SPRI purification or column purification.

16. (e) amplifying the product of step (d) by PCR using primers that hybridize to the first region and the eighth region, the primers comprising index sequences for next generation sequencing. The method of any one of claims 1 to 15, further comprising:

17. 17. The method of claim 16, further comprising purifying the product of step (e).

18. 18. The method of claim 17, wherein purifying comprises SPRI purification or column purification.

19. (f) performing high throughput DNA sequencing of the products of step (e). The method of any one of claims 16 to 18, further comprising:

20. 20. The method of claim 19, wherein high throughput DNA sequencing comprises next generation sequencing.

21. The method of any one of claims 1 to 20, wherein the first target genomic DNA region and the second target genomic DNA region are on opposite strands of the genomic DNA.

22. 22. The method of any one of claims 1 to 21, wherein the first target genomic DNA region and the second target genomic DNA region are separated by 40 nucleotides to 500 nucleotides.

23. 23. The method of any one of claims 1 to 22, wherein step (b) comprises an extension time of about 30 minutes.

24. The method of any one of claims 1 to 23, wherein step (c) comprises an extension time of about 30 seconds.

25. 25. The method of any one of claims 1 to 24, wherein step (d) comprises an extension time of about 30 minutes.

26. 1. A method for quantifying the frequency of overcopy (FEC) of at least one target gene, comprising: (a) obtaining a genomic DNA sample; (b) preparing the genomic DNA for high throughput sequencing according to the method of any one of claims 1 to 25, wherein the sequences of the fourth region, the seventh region, and the tenth region hybridize to the at least one target gene, and each first oligonucleotide hybridizing to a target gene has a unique third region compared to each other first oligonucleotide hybridizing to the same target gene; (c) performing high throughput sequencing according to the method of claim 19; (d) calculating the FEC for the at least one target gene based on the sequence information obtained in step (c); The method comprising:

27. 27. The method of claim 26, wherein the method is for quantifying the FEC for a set of target genes, the set of target genes comprising between 2 and 1000 target genes.

28. 28. The method of claim 26 or 27, wherein step (b) is carried out using a first population of oligonucleotides, a second population of oligonucleotides, and a fifth population of oligonucleotides, a portion of each of the first, second, and fifth populations of oligonucleotides comprising a fourth, seventh, and tenth region, respectively, that is complementary to one of the set of target genes.

29. 29. The method of any one of claims 26 to 28, wherein each of the fourth, seventh and tenth regions comprises a sequence that is found only once in the human genome.

30. 30. The method of any one of claims 26 to 29, wherein step (b) is carried out using a first oligonucleotide, a second oligonucleotide, and a fifth oligonucleotide comprising a fourth, a seventh, and a tenth region, respectively, that are complementary to a reference gene.

31. 31. The method of any one of claims 26 to 30, wherein step (b) prepares a portion of each target or reference gene for high-throughput sequencing, said portion being between 40 nucleotides and 500 nucleotides in length.

32. The FEC is as follows: [0010] The method according to any one of claims 26 to 31, wherein said method is defined as

33. Step (d) (i) aligning NGS reads to the targeted portion of each target gene and grouping the NGS reads into subgroups based on the loci to which they align; (ii) classifying the NGS reads at each locus based on their UMI sequences such that all NGS reads carrying the same UMI sequence are grouped as one UMI family; (iii) removing UMI families resulting from PCR or NGS errors; (iv) counting the number of unique UMI sequences at each locus; and (v) calculating the FEC for each locus in each target gene and each reference gene based on the number of unique UMI sequences; The method according to any one of claims 26 to 32, comprising:

34. 34. The method of claim 33, wherein step (d)(iii) comprises removing UMI sequences that do not fit the UMI degenerate base design.

35. 35. The method of claim 33 or 34, wherein step (d)(iii) comprises removing UMI families having a UMI family size smaller than Fmin, the UMI family size being the number of reads carrying the same UMI, and Fmin being between 2 and 20.

36. 36. The method of any one of claims 33 to 35, wherein step (d)(iv) comprises removing UMI sequences that differ by only one or two bases from another UMI sequence that has a larger family size.

37. The FEC is as follows: [0025] is defined as: [0030] is the sum of the number of unique UMIs for all or a portion of the target loci, u is the number of loci under consideration, u is less than or equal to the total number of loci in the target locus, [0045] 37. The method of any one of claims 26 to 36, wherein v is the sum of the number of unique UMIs for all or a portion of the reference loci, v is the number of loci considered for a reference, v is less than or equal to the total number of loci in said reference, w is the number of references considered, w is less than or equal to the total number of references, and k is determined by experimental calibration.

38. The method of any one of claims 26 to 37, wherein said FEC is used to identify the copy number variation (CNV) status of said target genes.

39. 1. A method for quantifying allelic ratios of different genetic identities for at least one target genomic locus, comprising: (a) obtaining a genomic DNA sample; (b) preparing the genomic DNA for high throughput sequencing according to the method of any one of claims 1 to 25, wherein the sequences of the fourth region, the seventh region, and the tenth region hybridize to the genomic DNA near the at least one target genomic locus, and each first oligonucleotide that hybridizes to the genomic DNA near a target genomic locus has a unique third region compared to each other first oligonucleotide that hybridizes to the genomic DNA near the same target genomic locus; (c) performing high throughput sequencing according to the method of claim 19; (d) calculating allele ratios of different genetic identities for the at least one target genomic locus based on the sequencing information obtained in step (c); The method comprising:

40. 40. The method of claim 39, wherein the method is for quantifying the allelic ratios of different genetic identities for a set of target genomic loci, wherein the set of target genomic loci comprises between 2 and 10,000 target genomic loci.

41. 41. The method of claim 39 or 40, wherein step (b) is carried out using a first population of oligonucleotides, a second population of oligonucleotides, and a fifth population of oligonucleotides, a portion of each of the first, second, and fifth populations of oligonucleotides comprising a fourth, seventh, and tenth region, respectively, that is complementary to the genomic DNA near at least one of the set of target genomic loci.

42. 42. The method of any one of claims 39 to 41, wherein each of the fourth, seventh, and tenth regions comprises a sequence that cannot hybridize to non-target regions of the genomic DNA under the conditions of step (b).

43. 43. The method of any one of claims 39 to 42, wherein each target genomic locus is between 40 nucleotides and 500 nucleotides in length.

44. Step (d) (i) aligning NGS reads to the targeted genomic loci and grouping the NGS reads into subgroups based on the loci to which they align; (ii) classifying the NGS reads at each locus based on their UMI sequences such that all NGS reads carrying the same UMI sequence are grouped as one UMI family; (iii) removing UMI families resulting from PCR or NGS errors; (iv) determining the genetic identity for each remaining UMI family; and (v) counting the number of unique UMI sequences at each locus; and (vi) calculating said allelic ratio; The method according to any one of claims 39 to 43, comprising:

45. 45. The method of claim 44, wherein step (d)(iii) comprises removing UMI sequences that do not fit the UMI degenerate base design.

46. 46. ​​The method of claim 44 or 45, wherein step (d)(iii) comprises removing UMI families having a UMI family size smaller than Fmin, the UMI family size being the number of reads carrying the same UMI, and Fmin being between 2 and 20.

47. 47. The method of any one of claims 44 to 46, wherein step (d)(iii) comprises removing UMI sequences that differ by only one or two bases from another UMI sequence that has a larger family size.

48. 48. The method of any one of claims 44 to 47, wherein step (d)(iv) comprises determining the genetic identity only if at least 70% of the reads in a UMI family are identical at the genetic locus of interest.

49. The allelic ratio is R 対立遺伝子 = N １ / N ２ where N １ is the number of unique UMIs for the first genetic identity, and N ２ The method of any one of claims 39 to 48, wherein x is the number of unique UMIs for the second genetic identity.

50. 49. The method of any one of claims 44 to 48, wherein step (d)(iv) comprises identifying a consensus sequence for each UMI family.

51. 51. The method of claim 50, wherein the consensus sequence is the sequence that occurs most frequently in the UMI family.

52. 52. The method of claim 50 or 51, further comprising comparing said consensus sequence to a wild-type sequence for said locus, thereby identifying mutations in said consensus sequence.

53. 53. The method of claim 52, further comprising calculating the variant allele frequency (VAF) of the identified mutation.

54. 54. The method of claim 53, wherein the VAF of the identified mutation is defined as the number of UMI families that have the mutation / total number of UMI families.

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