The Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, MD, USA.
State Key Laboratory of Genetic Engineering, Human Phenome Institute, School of Life Sciences and Shanghai Cancer Center, Fudan University, Shanghai, China.
Nat Biotechnol. 2021 Sep;39(9):1141-1150. doi: 10.1038/s41587-021-00994-5. Epub 2021 Sep 9.
Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.
精准肿瘤学的临床应用需要准确的检测方法，能够区分真正的肿瘤特异性突变和下一代测序（NGS）每个步骤中引入的错误。迄今为止，尚无批量测序研究解决跨站点可重复性的影响，也没有研究影响变异识别的生物学、技术和计算因素。在这里，我们报告了一项对配对肿瘤-正常细胞系中的体细胞突变进行的系统研究，以确定影响六个不同中心检测重现性和准确性的因素。使用全基因组测序（WGS）和全外显子组测序（WES），我们评估了不同样本类型的重现性，包括不同的起始量和肿瘤纯度，以及多种文库构建方案，然后使用九个生物信息学流程进行处理。我们发现，读长覆盖度和调用者都会影响 WGS 和 WES 的重现性，但 WES 的性能受到插入片段大小、基因组拷贝含量和全局不平衡评分（GIV；G>T/C>A）的影响。最后，考虑到文库制备方案、肿瘤含量、读长覆盖度和生物信息学流程，我们建议采取切实可行的措施，以提高癌症突变检测的 NGS 实验的重现性和准确性。
