Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing.
Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing.
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DOI：
10.1038/s41551-021-00713-0
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发表时间：
2021-07
影响因子：
28.1
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中科院分区：
文献类型：
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由于合成测序方法存在固有错误，等位基因分数低于 1% 的 DNA 序列变异很难通过测序进行检测和定量。尽管分子识别条形码可以通过下一代测序 (NGS) 检测变异等位基因频率 (VAF) 低至 0.1% 的突变，但需要超过 25,000 倍的测序深度，从而阻碍了在患者样本和大多数研究中使用的样本中高灵敏度地检测突变。在这里，我们表明，低频 DNA 变体可以在扩增后通过低深度多重 NGS 进行检测，中值扩增 300 倍，并使用聚合酶链式反应和合理设计的与变体结合的“阻断剂”寡核苷酸。使用 80 重 NGS panel 和 250x 测序深度，我们检测到 VAF 中的单核苷酸多态性低至 0.019%，并且 VAF 中人类细胞系的污染低至 0.07%。通过覆盖涉及黑色素瘤的 9 个基因的 145 个突变的 16 重 NGS 检测，我们在 19 个新鲜冷冻肿瘤活检样本中的 7 个样本中检测到了低 VAF 突变 (0.2-5%)，这表明肿瘤异质性可能显着高于之前认识的水平。低频 DNA 变异可通过聚合酶链式反应和合理设计的“阻断剂”寡核苷酸扩增后，通过低深度多重测序进行检测。
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 down to 0.1% in variant allele frequency (VAF) with next-generation sequencing (NGS), sequencing depths of over 25,000x 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 300-fold in median, with the polymerase chain reaction and rationally designed ‘blocker’ oligonucleotides that bind to the variants. Using an 80-plex NGS panel and 250x in sequencing depth, we detected single nucleotide polymorphisms down to 0.019% in VAF, and contamination in human cell lines down to 0.07% in VAF. 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 19 samples of freshly frozen tumour biopsies, suggesting that tumour heterogeneity could be significantly higher than previously recognized. Low-frequency DNA variants can be detected via low-depth multiplexed sequencing after their amplification via the polymerase chain reaction and rationally designed ‘blocker’ oligonucleotides.
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影响因子：
3.7
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Merker M;Kohl TA;Roetzer A;Truebe L;Richter E;Rüsch-Gerdes S;Fattorini L;Oggioni MR;Cox H;Varaine F;Niemann S
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Horbach SPJM;Halffman W
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通讯作者：
Colston, Bill W.
影响因子：
46.9
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通讯作者：
Alizadeh AA