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测试，以区分真正的癌症特异性突变和下一代测序 (NGS) 每一步引入的错误。迄今为止，还没有批量测序研究解决了跨位点重现性的影响，也没有解决影响变异识别的生物、技术和计算因素。在这里，我们报告了对配对肿瘤-正常细胞系中体细胞突变的系统询问，以确定影响六个不同中心检测再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），我们评估了具有不同输入量和肿瘤纯度的不同样本类型的重现性，以及多种文库构建方案，然后用九个生物信息学管道进行处理。我们发现读取覆盖率和调用者都会影响 WGS 和 WES 重现性，但 WES 性能受到插入片段大小、基因组拷贝内容和全局不平衡评分 (GIV; G > T/C > A) 的影响。最后，考虑到文库制备方案、肿瘤内容、读数覆盖率和生物信息学过程，我们建议采取可行的做法，以提高癌症突变检测 NGS 实验的可重复性和准确性。