From the Departments of Pathology and.
Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
Am J Clin Pathol. 2014 Jun;141(6):856-66. doi: 10.1309/AJCPMWGWGO34EGOD.
To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth.
We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA).
We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations.
Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts.
验证下一代测序（NGS）技术在临床诊断中的应用，并确定合适的读取深度。
我们使用 Ion Torrent Personal Genome Machine（Life Technologies，加利福尼亚州卡尔斯巴德）对 Ion AmpliSeq Cancer Hotspot Panel 中的 KRAS、BRAF 和 EGFR 基因进行了验证。
我们开发了一种统计模型，用于确定给定肿瘤细胞比例和功能基因组数量所需的读取深度。瓶颈可能是由于输入基因组太少造成的。通过使用 16 份福尔马林固定、石蜡包埋（FFPE）的无癌标本和 118 份已知突变状态的癌症标本，我们验证了下一代测序标准临床检测工作组推荐的六个传统分析性能特征。基线噪声与自发和 FFPE 诱导的 C:G→T:A 脱氨突变一致。
冗余的生物信息学管道是必不可少的，因为单个分析管道会产生假阴性和假阳性结果。NGS 足够强大，可以用于临床检测基因突变，但需要注意潜在的伪影。
