Characterization of background noise in capture-based targeted sequencing data.
Characterization of background noise in capture-based targeted sequencing data.
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DOI：
10.1186/s13059-017-1275-2
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发表时间：
2017-07-21
期刊：
影响因子：
12.3
通讯作者：
Park D
中科院分区：
文献类型：
--
作者：
Park G;Park JK;Shin SH;Jeon HJ;Kim NKD;Kim YJ;Shin HT;Lee E;Lee KH;Son DS;Park WY;Park D
靶向深度测序越来越多地被用于检测低等位基因部分变异；因此，至关重要的是，必须确定构成基线噪声并对检测施加实际限制的错误。在本研究中，我们系统地评估了在基于捕获的靶向测序过程的特定步骤中发生错误的程度。我们通过过滤掉低质量的碱基，然后分析剩余的背景噪声，去除了大多数测序伪像。通过认识到血浆DNA自然被片段化，其大小与单核小体DNA的大小相当，我们能够识别和表征与声剪切特定相关的错误。三分之二的C：G >； A：T误差和四分之一的C：G >； G：C误差归因于声学剪切过程中鸟嘌呤的氧化，并通过在不同剪切条件下进行的对比实验进一步验证了这一点。声波剪切步骤还导致A >； G和A >； T替换定位于剪切的dna片段的末端碱基，这表明这些错误可能与dna断裂有关。最后，混合选择步骤导致了其余C：G &> A：T错误的三分之一和C：G &> T错误的五分之一。这项研究的结果全面总结了靶向深度测序过程中产生的各种错误及其根本原因。这些信息对推动这种测序方法的技术改进将是非常有价值的，并可能增加未来用于低等位基因比例变异检测的靶向深度测序方法的使用。本文的在线版本(doi：10.1186/s13059-017-1275-2)包含补充材料，授权用户可以使用。
Targeted deep sequencing is increasingly used to detect low-allelic fraction variants; it is therefore essential that errors that constitute baseline noise and impose a practical limit on detection are characterized. In the present study, we systematically evaluate the extent to which errors are incurred during specific steps of the capture-based targeted sequencing process. We removed most sequencing artifacts by filtering out low-quality bases and then analyze the remaining background noise. By recognizing that plasma DNA is naturally fragmented to be of a size comparable to that of mono-nucleosomal DNA, we were able to identify and characterize errors that are specifically associated with acoustic shearing. Two-thirds of C:G > A:T errors and one quarter of C:G > G:C errors were attributed to the oxidation of guanine during acoustic shearing, and this was further validated by comparative experiments conducted under different shearing conditions. The acoustic shearing step also causes A > G and A > T substitutions localized to the end bases of sheared DNA fragments, indicating a probable association of these errors with DNA breakage. Finally, the hybrid selection step contributes to one-third of the remaining C:G > A:T and one-fifth of the C > T errors. The results of this study provide a comprehensive summary of various errors incurred during targeted deep sequencing, and their underlying causes. This information will be invaluable to drive technical improvements in this sequencing method, and may increase the future usage of targeted deep sequencing methods for low-allelic fraction variant detection. The online version of this article (doi:10.1186/s13059-017-1275-2) contains supplementary material, which is available to authorized users.
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影响因子：
46.9
作者：
通讯作者：
--
DOI：
10.1073/pnas.0507904102
发表时间：
2005-11-08
影响因子：
11.1
作者：
Diehl, F;Li, M;Vogelstein, B
通讯作者：
Vogelstein, B
影响因子：
14.9
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
通讯作者：
Getz G
影响因子：
14.9
作者：
Kim J;Mrksich M
通讯作者：
Mrksich M
影响因子：
4.6
作者：
Chung J;Son DS;Jeon HJ;Kim KM;Park G;Ryu GH;Park WY;Park D
通讯作者：
Park D