Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.
Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.
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DOI：
10.1186/s12864-016-3425-4
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发表时间：
2017-01-03
期刊：
影响因子：
4.4
通讯作者：
Wang Y
中科院分区：
文献类型：
--
作者：
Xu C;Nezami Ranjbar MR;Wu Z;DiCarlo J;Wang Y
在非常低的等位基因分数下以高准确度检测DNA突变将显著提高癌症患者精准医疗的有效性。为了通过下一代测序实现这一目标，研究人员需要一种检测方法，该方法1）在丰富的野生型DNA混合物中有效捕获含有罕见突变的DNA片段; 2）广泛地对DNA文库进行测序以深度覆盖; 3）以高准确度区分低水平的真实变体与扩增和测序错误。使用PCR引物的靶向富集为研究人员提供了一种方便的方法，可以使用台式测序仪对小而最相关的区域进行深度测序。分子条形码化（或索引化）提供了一种独特的解决方案，用于在分析上减少测序伪影。尽管在最近的文献中已经报道了不同的分子条形码化方案，但是大多数变体调用已经使用简单的定制脚本在有限的靶标上进行。通过结合先进的统计模型，可以显著提高条形码感知变异识别的分析性能。我们在这里提出了一种高效，简单和可扩展的富集方案，在多重PCR扩增中整合分子条形码。此外，我们还开发了smCounter，这是一个基于贝叶斯概率模型的开源、通用、条形码感知的变体调用程序。对smCounter进行优化，并在具有SNV和插入缺失的两个独立读段集上以5%和1%等位基因分数为基准。在编码区内以非常好的灵敏度和特异性调用变体。我们证明，我们可以准确地检测体细胞突变与等位基因分数低至1%的编码区使用我们的富集协议和变异呼叫。本文的在线版本（doi：10.1186/s12864-016-3425-4）包含补充材料，可供授权用户使用。
Detection of DNA mutations at very low allele fractions with high accuracy will significantly improve the effectiveness of precision medicine for cancer patients. To achieve this goal through next generation sequencing, researchers need a detection method that 1) captures rare mutation-containing DNA fragments efficiently in the mix of abundant wild-type DNA; 2) sequences the DNA library extensively to deep coverage; and 3) distinguishes low level true variants from amplification and sequencing errors with high accuracy. Targeted enrichment using PCR primers provides researchers with a convenient way to achieve deep sequencing for a small, yet most relevant region using benchtop sequencers. Molecular barcoding (or indexing) provides a unique solution for reducing sequencing artifacts analytically. Although different molecular barcoding schemes have been reported in recent literature, most variant calling has been done on limited targets, using simple custom scripts. The analytical performance of barcode-aware variant calling can be significantly improved by incorporating advanced statistical models. We present here a highly efficient, simple and scalable enrichment protocol that integrates molecular barcodes in multiplex PCR amplification. In addition, we developed smCounter, an open source, generic, barcode-aware variant caller based on a Bayesian probabilistic model. smCounter was optimized and benchmarked on two independent read sets with SNVs and indels at 5 and 1% allele fractions. Variants were called with very good sensitivity and specificity within coding regions. We demonstrated that we can accurately detect somatic mutations with allele fractions as low as 1% in coding regions using our enrichment protocol and variant caller. The online version of this article (doi:10.1186/s12864-016-3425-4) contains supplementary material, which is available to authorized users.
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影响因子：
4.4
作者：
Peng Q;Vijaya Satya R;Lewis M;Randad P;Wang Y
通讯作者：
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DOI：
10.1073/pnas.1208715109
发表时间：
2012-09-04
影响因子：
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影响因子：
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影响因子：
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作者：
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