Boyle Theresa A, Mondal Ashis K, Saeed-Vafa Daryoush, Ananth Sudha, Ahluwalia Pankaj, Kothapalli Ravi, Chaubey Alka, Roberts Evans, Qin Dahui, Magliocco Anthony M, Rojiani Amyn M, Kolhe Ravindra
Department of Pathology and Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, United States.
Department of Pathology, Medical College of Georgia at Augusta University, Augusta, GA, United States.
Front Genet. 2021 May 20;12:503830. doi: 10.3389/fgene.2021.503830. eCollection 2021.
We describe the clinical validation of a targeted DNA and RNA-based next-generation sequencing (NGS) assay at two clinical molecular diagnostic laboratories. This assay employs simultaneous DNA and RNA analysis of all coding exons to detect small variants (single-nucleotide variants, insertions, and deletions) in 148 genes, amplifications in 59 genes, and fusions and splice variants in 55 genes. During independent validations at two sites, 234 individual specimens were tested, including clinical formalin-fixed, paraffin-embedded (FFPE) tumor specimens, reference material, and cell lines. Samples were prepared using the Illumina TruSight Tumor 170 (TST170) kit, sequenced with Illumina sequencers, and the data were analyzed using the TST170 App. At both sites, TST170 had ≥98% success for ≥250× depth for ≥95% of covered positions. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection studies determined that inputs of ≥50 ng of DNA (with ≥3.3 ng/μl) and ≥50 ng RNA (minimum of 7 copies/ng) were optimal for high analytical sensitivity. The TST170 assay results were highly concordant with prior results using different methods across all variant categories. Optimization of nucleic acid extraction and DNA shearing, and quality control following library preparation is recommended to maximize assay success rates. In summary, we describe the validation of comprehensive and simultaneous DNA and RNA-based NGS testing using TST170 at two clinical sites.
我们描述了一种基于靶向DNA和RNA的新一代测序（NGS）检测方法在两个临床分子诊断实验室的临床验证情况。该检测方法同时对所有编码外显子进行DNA和RNA分析，以检测148个基因中的小变异（单核苷酸变异、插入和缺失）、59个基因中的扩增以及55个基因中的融合和剪接变异。在两个地点进行独立验证期间，共检测了234个个体样本，包括临床福尔马林固定、石蜡包埋（FFPE）肿瘤样本、参考材料和细胞系。样本使用Illumina TruSight Tumor 170（TST170）试剂盒制备，用Illumina测序仪进行测序，并使用TST170应用程序分析数据。在两个地点，对于≥95%的覆盖位置，TST170在≥250×深度时成功率≥98%。在等位基因频率≥5%时，变异检测准确且可重复。检测限研究确定，≥50 ng的DNA（≥3.3 ng/μl）和≥50 ng的RNA（至少7拷贝/ng）输入量对于高分析灵敏度是最佳的。TST170检测结果与之前使用不同方法在所有变异类别中的结果高度一致。建议优化核酸提取和DNA剪切，并在文库制备后进行质量控制，以最大限度提高检测成功率。总之，我们描述了在两个临床地点使用TST170对基于DNA和RNA的综合同步NGS检测进行的验证。
