多路复用FFPE扩增子测序技术和循环,游离DNA

体细胞突变的检测具有挑战性,因为肿瘤百分比含量的临床样本变量,并加剧了肿瘤的异质性。此外,循环cfDNA和FFPE样本通常是有限的数量,和FFPE样本也可以高度受损。为应对这些挑战,我们开发了一个单管,多路复用雇用了数以百计的引物扩增子测序方法对目标位点的放大生产就绪的库Illumina公司®测序。两步方法——多路复用PCR后10分钟适配器结扎-导致扩增子120 - 160 bp的长度,使放大和变体打来cfDNA-sized DNA片段或损坏FFPE DNA。使用这种技术,研制了一个肿瘤小组目标,56临床相关的突变基因。例如,抗EGFR在cfDNA针对使液体活检监测样本。面板设计包含单一外显子(例如BRAF)以及综合编码外显子覆盖整个基因(例如TP53),根据等位基因分布在每个目标基因。来验证这个面板,一群控制和临床样本pre-validated基因型进行了测试使用10 ng输入DNA。扩增子库MiSeq被qPCR量化和排序。校准和变体调用使用验证,进行公开可用的工具和手动确认。 Robust detection of 5% mutant frequency was observed for samples, and in performing spike-in experiments, the limit of detection was as low as 1% mutant frequency. The percent on-target bases and coverage uniformity were both >95%, where uniformity is defined as the percent bases covered at >20% of the mean coverage. These results indicate that this multiplexed amplicon panel is an excellent tool to assess multiple oncogenes in limiting clinical samples, enabling high throughput, cost effective NGS analysis.