Dataset Information

Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing.

ABSTRACT: DNA sequence variants with allele fractions below 1% are difficult to detect and quantify by sequencing owing to intrinsic errors in sequencing-by-synthesis methods. Although molecular-identifier barcodes can detect mutations with a variant-allele frequency (VAF) as low as 0.1% using next-generation sequencing (NGS), sequencing depths of over 25,000× are required, thus hampering the detection of mutations at high sensitivity in patient samples and in most samples used in research. Here we show that low-frequency DNA variants can be detected via low-depth multiplexed NGS after their amplification, by a median of 300-fold, using polymerase chain reaction and rationally designed 'blocker' oligonucleotides that bind to the variants. Using an 80-plex NGS panel and a sequencing depth of 250×, we detected single nucleotide polymorphisms with a VAF of 0.019% and contamination in human cell lines at a VAF as low as 0.07%. With a 16-plex NGS panel covering 145 mutations across 9 genes involved in melanoma, we detected low-VAF mutations (0.2-5%) in 7 out of the 19 samples of freshly frozen tumour biopsies, suggesting that tumour heterogeneity could be notably higher than previously recognized.

SUBMITTER: Song P

PROVIDER: S-EPMC9631981 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing.

Song Ping P Chen Sherry X SX Yan Yan Helen YH Pinto Alessandro A Cheng Lauren Y LY Dai Peng P Patel Abhijit A AA Zhang David Yu DY

Nature biomedical engineering 20210503 7

DNA sequence variants with allele fractions below 1% are difficult to detect and quantify by sequencing owing to intrinsic errors in sequencing-by-synthesis methods. Although molecular-identifier barcodes can detect mutations with a variant-allele frequency (VAF) as low as 0.1% using next-generation sequencing (NGS), sequencing depths of over 25,000× are required, thus hampering the detection of mutations at high sensitivity in patient samples and in most samples used in research. Here we show t ...[more]

PMID: 33941896

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Dataset Information

Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing.

Publications

Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing.

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