Lin Hsin-Fu, Chien Pei-Miao, Cheng Chinyi, Yuan Tzu-Hang, Wang Yu-Bin, Chen Pei-Lung, Chen Chien-Yu, Huang Jia-Hsin, Hsu Jacob Shujui
Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, Taiwan.
Graduate Institute of Medical Genomics and Proteomics, College of Medicine, National Taiwan University, Taipei, Taiwan; Taiwan AI Labs, Taipei, Taiwan.
J Mol Diagn. 2025 Aug;27(8):768-782. doi: 10.1016/j.jmoldx.2025.04.012. Epub 2025 Jun 13.
Evaluating robustness of somatic mutation detections is essential when using whole-exome sequencing (WES) for treatment decision-making. A comprehensive evaluation was conducted using tumor WES from the US Food and Drug Administration-led Sequencing Quality Control Phase 2 project, in which multiple library kits sequenced identical DNA materials across three laboratories to benchmark analytical validity. These workflows included various read aligner (BWA, Bowtie2, DRAGEN-Aligner, DRAGMAP, and HISAT2) and mutation caller (Mutect2, TNscope, DRAGEN-Caller, and DeepVariant) combinations. The results revealed that DRAGEN exhibited superior performance, achieving mean F1 scores of 0.966 and 0.791 for single-nucleotide variant and insertion/deletion detection, respectively. Among open-source software, BWA Mutect2 and HISAT2 Mutect2 combinations showed the highest mean F1 scores for single-nucleotide variant (0.949) and insertion/deletion (0.722), respectively. The analyses indicated that high-quality data can be analyzed as having worse results, and vice versa. Evaluations of Catalog of Somatic Mutations in Cancer reported mutations unveiled discrepancies across enrichment kits. Integrated DNA Technologies enrichment kits showed a higher false-negative rate, whereas Agilent WES kits tended to miss mutations in CBL and IDH1, and Roche library kits tended to miss the mutations in PIK3CB. Sentieon TNscope tended to underestimate tumor mutation burden and overlook FLT3:c.G1879A for cytarabine resistance in leukemia and MAP2K1:c.G199A for BRAF inhibitors in melanoma. The findings highlight the importance of robust bioinformatic analysis in guiding clinical decision-making.
在使用全外显子组测序（WES）进行治疗决策时，评估体细胞突变检测的稳健性至关重要。我们利用美国食品药品监督管理局主导的测序质量控制第二阶段项目中的肿瘤WES进行了全面评估，在该项目中，多个文库试剂盒在三个实验室对相同的DNA材料进行测序，以基准分析有效性。这些工作流程包括各种读段比对器（BWA、Bowtie2、DRAGEN - Aligner、DRAGMAP和HISAT2）和突变检测工具（Mutect2、TNscope、DRAGEN - Caller和DeepVariant）的组合。结果显示，DRAGEN表现出卓越的性能，在单核苷酸变异和插入/缺失检测方面的平均F1分数分别达到0.966和0.791。在开源软件中，BWA Mutect2和HISAT2 Mutect2组合在单核苷酸变异（0.949）和插入/缺失（0.722）方面分别显示出最高的平均F1分数。分析表明，高质量的数据可能被分析出较差的结果，反之亦然。对癌症体细胞突变目录报告的突变评估揭示了不同富集试剂盒之间的差异。Integrated DNA Technologies富集试剂盒显示出较高的假阴性率，而安捷伦WES试剂盒往往会遗漏CBL和IDH1中的突变，罗氏文库试剂盒则往往会遗漏PIK3CB中的突变。Sentieon TNscope往往会低估肿瘤突变负担，并忽略白血病中阿糖胞苷耐药相关的FLT3:c.G1879A突变以及黑色素瘤中BRAF抑制剂相关的MAP2K1:c.G199A突变。这些发现凸显了稳健的生物信息学分析在指导临床决策中的重要性。
