Luthra Rajyalakshmi, Patel Keyur P, Routbort Mark J, Broaddus Russell R, Yau Jonathan, Simien Crystal, Chen Wei, Hatfield David Z, Medeiros L Jeffrey, Singh Rajesh R
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas.
J Mol Diagn. 2017 Mar;19(2):255-264. doi: 10.1016/j.jmoldx.2016.09.011. Epub 2016 Dec 23.
Clinical next-generation sequencing (NGS) assay choice requires careful consideration of panel size, inclusion of appropriate markers, ability to detect multiple genomic aberration types, compatibility with low quality and quantity of nucleic acids, and work flow feasibility. Herein, in a high-volume clinical molecular diagnostic laboratory, we have validated a targeted high-multiplex PCR-based NGS panel (OncoMine Comprehensive Assay) coupled with high-throughput sequencing using Ion Proton sequencer for routine screening of solid tumors. The panel screens 143 genes using low amounts of formalin-fixed, paraffin-embedded DNA (20 ng) and RNA (10 ng). A large cohort of 121 tumor samples representing 13 tumor types and 6 cancer cell lines was used to assess the capability of the panel to detect 148 single-nucleotide variants, 49 insertions or deletions, 40 copy number aberrations, and a subset of gene fusions. High levels of analytic sensitivity and reproducibility and robust detection sensitivity were observed. Furthermore, we demonstrated the critical utility of sequencing paired normal tissues to improve the accuracy of detecting somatic mutations in a background of germline variants. We also validated use of the Ion Chef automated bead templating and chip loading system, which represents a major work flow improvement. In summary, we present data establishing the OncoMine Comprehensive Assay-Ion Proton platform to be well suited for implementation as a routine clinical NGS test for solid tumors.
临床下一代测序（NGS）检测方法的选择需要仔细考虑面板大小、适当标志物的纳入、检测多种基因组畸变类型的能力、与低质量和低数量核酸的兼容性以及工作流程的可行性。在此，在一个高产量的临床分子诊断实验室中，我们已经验证了一种基于靶向高多重PCR的NGS面板（OncoMine综合检测法），并结合使用Ion Proton测序仪进行高通量测序，用于实体瘤的常规筛查。该面板使用少量福尔马林固定、石蜡包埋的DNA（20 ng）和RNA（10 ng）对143个基因进行筛查。使用代表13种肿瘤类型的121个肿瘤样本的大型队列和6种癌细胞系来评估该面板检测148个单核苷酸变异、49个插入或缺失、40个拷贝数畸变以及一部分基因融合的能力。观察到了高水平的分析灵敏度和重现性以及强大的检测灵敏度。此外，我们证明了对配对正常组织进行测序以提高在种系变异背景下检测体细胞突变准确性的关键效用。我们还验证了Ion Chef自动磁珠模板制备和芯片加载系统的使用，这代表了工作流程的重大改进。总之，我们提供的数据表明OncoMine综合检测法 - Ion Proton平台非常适合作为实体瘤的常规临床NGS检测方法来实施。
