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首页> 外文期刊>The Journal of molecular diagnostics: JMD >A Targeted High-Throughput Next-Generation Sequencing Panel for Clinical Screening of Mutations, Gene Amplifications, and Fusions in Solid Tumors
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A Targeted High-Throughput Next-Generation Sequencing Panel for Clinical Screening of Mutations, Gene Amplifications, and Fusions in Solid Tumors

机译：靶向高通量的下一代测序面板，用于临床筛查突变，基因扩增和实体肿瘤的融合

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摘要

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 tow 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.
机译：临床下一代测序（NGS）测定选择需要仔细考虑面板大小，包含适当的标记，检测多种基因组畸变类型的能力，与低质量和核酸量的相容性，以及工作流动性。这里，在大批量临床分子诊断实验室中，我们已经验证了使用离子质子测序仪的高通量测序的靶向高多重PCR的NGS面板（oncomeSine综合测定），用于使用离子质子测序仪进行实体瘤的常规筛选。使用筛选量的福尔马林固定的，石蜡嵌入DNA（20ng）和RNA（10ng）的面板筛选143基因。使用代表13种肿瘤类型和6种癌细胞系的大队列的121颗肿瘤样本来评估面板检测148个单核苷酸变体，49个插入或缺失，40拷贝数像差和基因融合子集的能力。观察到高水平的分析敏感性和再现性和鲁棒检测灵敏度。此外，我们证明了测序配对正常组织的关键效用，以提高种系变体背景中检测体细胞突变的准确性。我们还经过了使用离子厨师自动珠子模板和芯片装载系统的使用，这代表了主要的工作流程改进。总之，我们呈现建立oncomeine综合测定离子平台的数据，以适应于实施的实体肿瘤的常规临床NGS试验。

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