Quantitative amplicon sequencing for multiplexed copy number variation detection and allele ratio quantitation

A method for performing copy number variant (CNV) detection and/or allele ratio quantitation of at least ten target genomic loci each having three target genomic DNA regions for high-throughput sequencing, the method comprising:

• (a) obtaining a genomic DNA sample;
• (b) amplifying at least a portion of the genomic DNA sample by performing two cycles of multiplex PCR using:
• (i) a population of first oligonucleotides each comprising, from 5′ to 3′, a first region, a second region having a length between 1 and 50 nucleotides, a third region comprising at least four degenerate nucleotides, and a fourth region comprising a sequence that is complementary to a first target genomic DNA region for one of the target genomic loci;
• and
• (ii) a population of second oligonucleotides each comprising, from 5′ to 3′, a fifth region, a sixth region having a length between 1 and 50 nucleotides, and a seventh region comprising a sequence that is complementary to a second target genomic DNA region for one of the target genomic loci;
• (c) amplifying a product of step (b) by performing at least three cycles of PCR with an annealing temperature that is 0-10° C. higher than an annealing temperature used in step (b) and using:
• (i) a third oligonucleotide comprising a sequence that is able to hybridize to the reverse complement of at least a portion of the first region;
• and
• (ii) a fourth oligonucleotide comprising a sequence that is able to hybridize to the reverse complement of at least a portion of the fifth region;
• (d) amplifying a product of step (c) by performing at least one cycle of PCR using a population of fifth oligonucleotides each comprising, from 5′ to 3′, an eighth region, a ninth region having a length between 1 and 50 nucleotides, and a tenth region comprising a sequence that is complementary to a third target genomic DNA region for one of the target genomic loci, 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 for each of the target genomic loci, thereby producing amplicons;
• (e) sequencing the amplicons by performing quantitative next-generation sequencing (qNGS) to obtain sequencing reads;
• and
• (f) counting the sequencing reads to perform CNV detection and/or allele ratio quantitation for the at least 10 target genomic regions.