Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation.
Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation.
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DOI：
10.1101/gr.147686.112
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发表时间：
2013-05
期刊：
影响因子：
7
通讯作者：
Shendure J
中科院分区：
文献类型：
--
作者：
Hiatt JB;Pritchard CC;Salipante SJ;O'Roak BJ;Shendure J
细胞群遗传异质性的检测和量化对于从微生物进化到人类癌症遗传学等各个领域都至关重要。然而，尽管与大规模并行测序相关的成本和通量有所提高，但可靠地检测给定 DNA 样本中相对丰度较低的突变仍然具有挑战性。在这里，我们描述了 smMIP，这是一种将单分子标记与多重靶向捕获相结合的检测方法，可实现低频或亚克隆变异的实用且高度灵敏的检测。为了证明该方法的潜力，我们同时对 8 个细胞系和 45 个临床癌症样本中的 33 个具有临床信息的癌症基因进行了重新测序。单分子标记促进了极其准确的共识调用，细胞系中的估计每碱基错误率为 8.4 × 10−6 ，临床样本中的每碱基错误率为 2.6 × 10−5 。单分子共有碱基识别中的假阳性突变表现出主要与 DNA 损伤一致的模式，包括 8-氧代鸟嘌呤和胞嘧啶的自发脱氨基。根据细胞系样本的混合实验，频率高于 1% 的突变的敏感性为 83%，没有假阳性。在临床信息位点，我们发现了 7 个低频点突变 (0.2%–4.7%)，包括 BRAF p.V600E（黑色素瘤，0.2% 替代等位基因频率）、KRAS p.G12V（肺，0.6%）、JAK2 p.V617F（黑色素瘤、结肠、两肺，0.3%–1.4%）和 NRAS p.Q61R （冒号，4.7%）。我们预计 smMIP 将作为一种实用且有效的方法被广泛采用，用于在研究和临床环境中准确检测低频突变。
The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10−6 in cell lines and 2.6 × 10−5 in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%–4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%–1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings.
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影响因子：
64.8
作者：
Ernst, Jason;Kheradpour, Pouya;Mikkelsen, Tarjei S.;Shoresh, Noam;Ward, Lucas D.;Epstein, Charles B.;Zhang, Xiaolan;Wang, Li;Issner, Robbyn;Coyne, Michael;Ku, Manching;Durham, Timothy;Kellis, Manolis;Bernstein, Bradley E.
通讯作者：
Bernstein, Bradley E.
使用下一代靶向重新取样对稀有突变的超敏感检测。
影响因子：
14.9
作者：
Flaherty P;Natsoulis G;Muralidharan O;Winters M;Buenrostro J;Bell J;Brown S;Holodniy M;Zhang N;Ji HP
通讯作者：
Ji HP
影响因子：
48
作者：
Hiatt, Joseph B.;Patwardhan, Rupali P.;Turner, Emily H.;Lee, Choli;Shendure, Jay
通讯作者：
Shendure, Jay
影响因子：
10.3
作者：
Lin, Jingrong;Takata, Minoru;Saida, Toshiaki
通讯作者：
Saida, Toshiaki
影响因子：
14.9
作者：
Casbon JA;Osborne RJ;Brenner S;Lichtenstein CP
通讯作者：
Lichtenstein CP