Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation.
Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation.
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DOI：
10.1093/nar/gks1443
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发表时间：
2013-04-01
影响因子：
14.9
通讯作者：
Getz G
中科院分区：
文献类型：
--
作者：
Costello M;Pugh TJ;Fennell TJ;Stewart C;Lichtenstein L;Meldrim JC;Fostel JL;Friedrich DC;Perrin D;Dionne D;Kim S;Gabriel SB;Lander ES;Fisher S;Getz G
随着研究人员开始探索深度覆盖测序数据以获得越来越罕见的突变和亚克隆事件，下一代测序（NGS）实验室方法的保真度将变得越来越重要。虽然测序和聚合酶链反应（PCR）的错误率有据可查，但DNA提取和其他文库制备步骤可能对下游序列完整性产生的影响尚未得到彻底评估。在这里，我们描述了新的C> A/G> T颠换伪像的发现，发现在靶向捕获数据中的低等位基因分数。诸如测序仪读取方向和在肿瘤和正常样品中的存在的特征强烈地指示非生物机制。我们确定的来源为DNA的氧化过程中含有反应性污染物的提取过程中的样品中的声剪切。我们展示了DNA剪切过程中8-氧代鸟嘌呤（8-oxoG）损伤的产生，提出了检测测序数据中氧化的分析工具，并提出了通过引入抗氧化剂来减少DNA氧化的方法。此外，提出了信息学方法来自信地从测序数据集中过滤这些伪影。虽然只在受影响样本中的低百分比读数中看到，但这些伪影可能对自信地调用罕见突变的能力产生严重的有害影响，消除其他可能的伪影来源应该成为研究界的优先事项。
As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.
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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：
4.8
作者：
Margolin, Yelena;Shafirovich, Vladimir;Dedon, Peter C.
通讯作者：
Dedon, Peter C.
影响因子：
4.1
作者：
Kennedy, LJ;Moore, K;Dedon, PC
通讯作者：
Dedon, PC
影响因子：
7.4
作者：
FUCIARELLI, AF;SISK, EC;MILLER, DL
通讯作者：
MILLER, DL
影响因子：
64.8
作者：
Berger, Michael F.;Hodis, Eran;Heffernan, Timothy P.;Deribe, Yonathan Lissanu;Lawrence, Michael S.;Protopopov, Alexei;Ivanova, Elena;Watson, Ian R.;Nickerson, Elizabeth;Ghosh, Papia;Zhang, Hailei;Zeid, Rhamy;Ren, Xiaojia;Cibulskis, Kristian;Sivachenko, Andrey Y.;Wagle, Nikhil;Sucker, Antje;Sougnez, Carrie;Onofrio, Robert;Ambrogio, Lauren;Auclair, Daniel;Fennell, Timothy;Carter, Scott L.;Drier, Yotam;Stojanov, Petar;Singer, Meredith A.;Voet, Douglas;Jing, Rui;Saksena, Gordon;Barretina, Jordi;Ramos, Alex H.;Pugh, Trevor J.;Stransky, Nicolas;Parkin, Melissa;Winckler, Wendy;Mahan, Scott;Ardlie, Kristin;Baldwin, Jennifer;Wargo, Jennifer;Schadendorf, Dirk;Meyerson, Matthew;Gabriel, Stacey B.;Golub, Todd R.;Wagner, Stephan N.;Lander, Eric S.;Getz, Gad;Chin, Lynda;Garraway, Levi A.
通讯作者：
Garraway, Levi A.