Asuragen, Inc., Austin, Texas, USA.
J Mol Diagn. 2013 Mar;15(2):234-47. doi: 10.1016/j.jmoldx.2012.11.006. Epub 2013 Jan 13.
Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.
将高度复杂的技术，如下一代测序（NGS），应用于常规临床实践需要与常见的肿瘤活检类型兼容，如福尔马林固定、石蜡包埋（FFPE）和细针穿刺标本，并需要为平台、对照和数据分析管道提供验证指标。在这项研究中，使用两步 PCR 富集工作流程来评估 16 个癌基因中的 540 个已知与癌症相关的变异，以便在成熟的（Illumina GAIIx）或新兴的（Ion Torrent PGM）NGS 平台上对肿瘤样本进行高深度测序。结果表明，与细胞系 DNA 相比，FFPE 中变异检测的背景噪声升高了约两倍。优化了生物信息学算法以适应这种背景。对 38 个残留的临床结直肠癌 FFPE 标本和 10 个甲状腺细针穿刺标本的变异进行了跨多个癌症基因的比较，与 Sanger 测序相比，准确率为 96.1%（95%CI，96.1%至 99.3%），与分析灵敏度为 1%的突变检测的替代方法相比，准确率为 99.6%（95%CI，97.9%至 99.9%）。在所有匹配区域中，48 个样本中有 45 个在两个 NGS 平台之间是一致的，三个不一致的结果各自仅占读取数的<10%。因此，使用不同平台对真实肿瘤标本中的靶向癌基因进行 NGS 可以满足对无偏和高度敏感的突变检测的未满足需求，并可以加速基础和临床癌症研究。
