Leeds Institute of Cancer and Pathology, St James's University Hospital, University of Leeds, Leeds, West Yorkshire, LS9 7TF, England.
Hum Mutat. 2013 Oct;34(10):1432-8. doi: 10.1002/humu.22365. Epub 2013 Jul 11.
Current methods for resolving genetically distinct subclones in tumor samples require somatic mutations to be clustered by allelic frequencies, which are determined by applying a variant calling program to next-generation sequencing data. Such programs were developed to accurately distinguish true polymorphisms and somatic mutations from the artifactual nonreference alleles introduced during library preparation and sequencing. However, numerous variant callers exist with no clear indication of the best performer for subclonal analysis, in which the accuracy of the assigned variant frequency is as important as correctly indicating whether the variant is present or not. Furthermore, sequencing depth (the number of times that a genomic position is sequenced) affects the ability to detect low-allelic fraction variants and accurately assign their allele frequencies. We created two synthetic sequencing datasets, and sequenced real KRAS amplicons, with variants spiked in at specific ratios, to assess which caller performs best in terms of both variant detection and assignment of allelic frequencies. We also assessed the sequencing depths required to detect low-allelic fraction variants. We found that VarScan2 performed best overall with sequencing depths of 100×, 250×, 500×, and 1,000× required to accurately identify variants present at 10%, 5%, 2.5%, and 1%, respectively.
当前，在肿瘤样本中解析遗传上不同亚克隆的方法需要根据等位基因频率对体细胞突变进行聚类，这是通过将变异调用程序应用于下一代测序数据来确定的。此类程序是为了准确区分真实的多态性和体细胞突变与文库制备和测序过程中引入的人为非参考等位基因而开发的。然而，存在许多变体调用者，对于亚克隆分析，没有明确表明哪种变体调用者表现最好，在亚克隆分析中，分配的变异频率的准确性与正确指示变异是否存在同样重要。此外，测序深度（基因组位置被测序的次数）会影响检测低等位基因分数变体并准确分配其等位基因频率的能力。我们创建了两个合成测序数据集，并对具有特定比例插入变体的真实 KRAS 扩增子进行了测序，以评估在变体检测和等位基因频率分配方面哪种调用者表现最好。我们还评估了检测低等位基因分数变体所需的测序深度。我们发现，VarScan2 在测序深度为 100×、250×、500×和 1000×时表现最佳，分别需要准确识别分别存在于 10%、5%、2.5%和 1%的变体。
