背景:靶向下一代测序(NGS)是一种强大且合适的方法,可全面鉴定肿瘤中多种类型的变体。基于RNA的NGS在精准肿瘤学中发挥着越来越重要的作用。基于DNA和RNA的平行和顺序方法都很昂贵,繁重的,并且有很长的周转时间,这在临床实践中可能是不切实际的。一个流线型的,在临床实践中迫切需要统一的基于DNA和RNA的NGS方法。
方法:设计了一种DNA/RNA共杂交捕获测序(DRCC-Seq)方法,用于在单个管中捕获预捕获DNA和RNA文库,并将其转化为一个NGS文库。通过一组参考标准和临床样品评估DRCC-Seq方法的性能。
结果:平均深度,DNA数据比率,捕获比,DNA面板数据的目标覆盖率250(×)与RNA探针比例的增加呈负相关。SNV,indels,融合,和MSI状态不受RNA探针比例的影响,但是目标基因的拷贝数高于标准材料中的预期,使用D:R(1:2)和D:R(1:4)探针面板发现了许多意想不到的基因扩增。使用DRCC-Seq方法,组合探针组中DNA和RNA探针的最佳比例为1:1。DRCC-Seq方法对于检测参考标准和真实世界临床样品中的多种类型的变体是可行和可靠的。
结论:DRCC-Seq方法更具成本效益,与平行或顺序靶向DNANGS和RNANGS相比,周转时间更短,劳动力需求更低。在临床实践中同时识别DNA和RNA水平的多种遗传变异是可行的。
BACKGROUND: Targeted next-generation sequencing (NGS) is a powerful and suitable approach to comprehensively identify multiple types of variants in tumors. RNA-based NGS is increasingly playing an important role in precision oncology. Both parallel and sequential DNA- and RNA-based approaches are expensive, burdensome, and have long turnaround times, which can be impractical in clinical practice. A streamlined, unified DNA- and RNA-based NGS approach is urgently needed in clinical practice.
METHODS: A DNA/RNA co-hybrid capture sequencing (DRCC-Seq) approach was designed to capture pre-capture DNA and RNA libraries in a single tube and convert them into one NGS library. The performance of the DRCC-Seq approach was evaluated by a panel of reference standards and clinical samples.
RESULTS: The average depth, DNA data ratio, capture ratio, and target coverage 250 (×) of the DNA panel data had a negative correlation with an increase in the proportion of RNA probes. The SNVs, indels, fusions, and MSI status were not affected by the proportion of RNA probes, but the copy numbers of the target genes were higher than expected in the standard materials, and many unexpected gene amplifications were found using D:R (1:2) and D:R (1:4) probe panels. The optimal ratio of DNA and RNA probes in the combined probe panel was 1:1 using the DRCC-Seq approach. The DRCC-Seq approach was feasible and reliable for detecting multiple types of variants in reference standards and real-world clinical samples.
CONCLUSIONS: The DRCC-Seq approach is more cost-effective, with a shorter turnaround time and lower labor requirements than either parallel or sequential targeted DNA NGS and RNA NGS. It is feasible to identify multiple genetic variations at the DNA and RNA levels simultaneously in clinical practice.