DNA测序
生物
寡核苷酸
聚合酶链反应
冷PCR
分子生物学
多重位移放大
遗传学
深度测序
数字聚合酶链反应
DNA
基因
突变
点突变
基因组
DNA提取
作者
Ping Song,Sherry X. Chen,Yan Helen Yan,Alessandro Pinto,Lauren Y. Cheng,Peng Dai,Abhijit A. Patel,David Y. Zhang
标识
DOI:10.1038/s41551-021-00713-0
摘要
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.
科研通智能强力驱动
Strongly Powered by AbleSci AI