PDF(Pubmed)
Abstract：
Lung cancer is a paradigm for a genetically driven tumor. A variety of drugs were developed targeting specific biomarkers requiring testing for tumor genetic alterations in relevant biomarkers. Different next-generation sequencing technologies are available for library generation: 1) anchored multiplex-, 2) amplicon based- and 3) hybrid capture-based-PCR. Anchored multiplex PCR-based sequencing was investigated for routine molecular testing within the national Network Genomic Medicine Lung Cancer (nNGM). Four centers applied the anchored multiplex ArcherDX-Variantplex nNGMv2 panel to re-analyze samples pre-tested during routine diagnostics. Data analyses were performed by each center and compiled centrally according to study design. Pre-defined standards were utilized, and panel sensitivity was determined by dilution experiments. nNGMv2 panel sequencing was successful in 98.9% of the samples (N = 90). With default filter settings, all but two potential MET exon 14 skipping variants were identified at similar allele frequencies. Both MET variants were found with an adapted calling filter. Three additional variants (KEAP1, STK11, TP53) were called that were not identified in pre-testing analyses. Only total DNA amount but not a qPCR-based DNA quality score correlated with average coverage. Analysis was successful with a DNA input as low as 6.25 ng. Anchored multiplex PCR-based sequencing (nNGMv2) and a sophisticated user-friendly Archer-Analysis pipeline is a robust and specific technology to detect tumor genetic mutations for precision medicine of lung cancer patients.
摘要：
肺癌是基因驱动肿瘤的范例。开发了针对特定生物标志物的多种药物，需要测试相关生物标志物中的肿瘤遗传改变。不同的下一代测序技术可用于文库生成：1)锚定多路复用-，2)基于扩增子和3)基于杂交捕获的PCR。在国家网络基因组医学肺癌（nNGM）中，对基于锚定多重PCR的测序进行了常规分子检测研究。四个中心应用了锚定的多重ArcherDX-VariantplexnNGMv2面板，以重新分析在常规诊断期间预先测试的样品。每个中心进行数据分析，并根据研究设计进行集中汇编。使用了预定义的标准，通过稀释实验确定面板灵敏度。nNGMv2组测序在98.9%的样品中成功(N=90)。使用默认过滤器设置，在相似的等位基因频率下鉴定出除了两个潜在的MET外显子14跳跃变异体之外的所有变异体.发现两种MET变体都具有适应的调用过滤器。三个另外的变体(KEAP1、STK11、TP53)被称为未在预测试分析中鉴定。仅总DNA量而不是基于qPCR的DNA质量评分与平均覆盖率相关。用低至6.25ng的DNA输入分析是成功的。基于锚定多重PCR的测序（nNGMv2）和复杂的用户友好的Archer-Analysis流程是一种强大而特定的技术，可检测肺癌患者的肿瘤基因突变。
