Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.
Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic.
Folia Biol (Praha). 2024;70(1):62-73. doi: 10.14712/fb2024070010062.
Germline DNA testing using the next-gene-ration sequencing (NGS) technology has become the analytical standard for the diagnostics of hereditary diseases, including cancer. Its increasing use places high demands on correct sample identification, independent confirmation of prioritized variants, and their functional and clinical interpretation. To streamline these processes, we introduced parallel DNA and RNA capture-based NGS using identical capture panel CZECANCA, which is routinely used for DNA analysis of hereditary cancer predisposition. Here, we present the analytical workflow for RNA sample processing and its analytical and diagnostic performance. Parallel DNA/RNA analysis allowed credible sample identification by calculating the kinship coefficient. The RNA capture-based approach enriched transcriptional targets for the majority of clinically relevant cancer predisposition genes to a degree that allowed analysis of the effect of identified DNA variants on mRNA processing. By comparing the panel and whole-exome RNA enrichment, we demonstrated that the tissue-specific gene expression pattern is independent of the capture panel. Moreover, technical replicates confirmed high reproducibility of the tested RNA analysis. We concluded that parallel DNA/RNA NGS using the identical gene panel is a robust and cost-effective diagnostic strategy. In our setting, it allows routine analysis of 48 DNA/RNA pairs using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) in a single run with sufficient coverage to analyse 226 cancer predisposition and candidate ge-nes. This approach can replace laborious Sanger confirmatory sequencing, increase testing turnaround, reduce analysis costs, and improve interpretation of the impact of variants by analysing their effect on mRNA processing.
基于下一代测序（NGS）技术的种系 DNA 检测已成为遗传性疾病（包括癌症）诊断的分析标准。随着其使用的增加，对正确的样本识别、优先变异的独立确认以及它们的功能和临床解释提出了很高的要求。为了简化这些过程，我们引入了基于平行 DNA 和 RNA 捕获的 NGS，使用相同的捕获面板 CZECANCA，该面板通常用于遗传性癌症易感性的 DNA 分析。在这里，我们介绍了 RNA 样本处理的分析工作流程及其分析和诊断性能。通过计算亲缘系数，平行 DNA/RNA 分析允许对可信的样本进行识别。基于 RNA 捕获的方法富集了大多数临床相关癌症易感性基因的转录靶标，程度足以分析鉴定的 DNA 变异对 mRNA 处理的影响。通过比较面板和全外显子 RNA 富集，我们证明了组织特异性基因表达模式独立于捕获面板。此外，技术重复证实了测试 RNA 分析的高重现性。我们得出结论，使用相同基因面板的平行 DNA/RNA NGS 是一种稳健且具有成本效益的诊断策略。在我们的环境中，它允许使用 NextSeq 500/550 Mid Output Kit v2.5（150 个循环）在单个运行中常规分析 48 对 DNA/RNA，具有足够的覆盖范围来分析 226 个癌症易感性和候选基因。这种方法可以替代繁琐的 Sanger 确认测序，提高测试周转时间，降低分析成本，并通过分析其对 mRNA 处理的影响来改善变异影响的解释。
