Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage
Systematic dissection of biases in whole-exome and whole-genome sequencing reveals major determinants of coding sequence coverage
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DOI：
10.1038/s41598-020-59026-y
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发表时间：
2020-02-06
影响因子：
4.6
通讯作者：
Predeus, Alexander V.
中科院分区：
文献类型：
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作者：
Barbitoff, Yury A.;Polev, Dmitrii E.;Predeus, Alexander V.
两种最广泛使用的重测序方法，全外显子组（WES）和全基因组（WGS）测序的优点和诊断有效性，经常被争论。WES在大规模重排序项目中占主导地位，因为它成本较低，数据存储和处理更容易。第三代测序方法和新型外显子组测序试剂盒的快速发展预示着需要一个强大的统计框架，允许新兴方法的信息和简单的性能比较。在我们的研究中，我们开发了一套统计工具来系统地评估几个现代WES平台以及无PCR WGS提供的编码区的覆盖率。我们发现了一个实质性的问题，在大多数以前发表的比较，没有考虑到映射的限制短读。使用回归分析和简单的机器学习，以及覆盖均匀性的几个新指标，我们分析了CDS覆盖率的主要决定因素的贡献。与普遍观点相反，现代WES中观察到的大部分偏差源于短读段和外显子组探针设计的可映射性限制，而不是序列组成。我们还鉴定了与人类外显子组的500kb区域相似的区域，这些区域不能使用短读技术有效地表征，并且在变异分析期间应受到特别关注。使用我们新的测序覆盖率指标，我们确定了WES和WGS性能的主要决定因素。总的来说，我们的研究指出了改进基于富集的方法和开发新方法的途径，这些方法将以最佳成本最大限度地发现变体。
Advantages and diagnostic effectiveness of the two most widely used resequencing approaches, whole exome (WES) and whole genome (WGS) sequencing, are often debated. WES dominated large-scale resequencing projects because of lower cost and easier data storage and processing. Rapid development of 3(rd) generation sequencing methods and novel exome sequencing kits predicate the need for a robust statistical framework allowing informative and easy performance comparison of the emerging methods. In our study we developed a set of statistical tools to systematically assess coverage of coding regions provided by several modern WES platforms, as well as PCR-free WGS. We identified a substantial problem in most previously published comparisons which did not account for mappability limitations of short reads. Using regression analysis and simple machine learning, as well as several novel metrics of coverage evenness, we analyzed the contribution from the major determinants of CDS coverage. Contrary to a common view, most of the observed bias in modern WES stems from mappability limitations of short reads and exome probe design rather than sequence composition. We also identified the similar to 500kb region of human exome that could not be effectively characterized using short read technology and should receive special attention during variant analysis. Using our novel metrics of sequencing coverage, we identified main determinants of WES and WGS performance. Overall, our study points out avenues for improvement of enrichment-based methods and development of novel approaches that would maximize variant discovery at optimal cost.