Department of Ophthalmology, University of Washington, Seattle, Washington, USA.
Roger and Karalis Johnson Retina Center, Seattle, Washington, USA.
JCI Insight. 2024 Sep 12;9(20):e183902. doi: 10.1172/jci.insight.183902.
Despite advances in sequencing technologies, a molecular diagnosis remains elusive in many patients with Mendelian disease. Current short-read clinical sequencing approaches cannot provide chromosomal phase information or epigenetic information without further sample processing, which is not routinely done and can result in an incomplete molecular diagnosis in patients. The ability to provide phased genetic and epigenetic information from a single sequencing run would improve the diagnostic rate of Mendelian conditions. Here, we describe targeted long-read sequencing of Mendelian disease genes (TaLon-SeqMD) using a real-time adaptive sequencing approach. Optimization of bioinformatic targeting enabled selective enrichment of multiple disease-causing regions of the human genome. Haplotype-resolved variant calling and simultaneous resolution of epigenetic base modification could be achieved in a single sequencing run. The TaLon-SeqMD approach was validated in a cohort of 18 individuals with previous genetic testing targeting 373 inherited retinal disease (IRD) genes, yielding the complete molecular diagnosis in each case. This approach was then applied in 2 IRD cases with inconclusive testing, which uncovered noncoding and structural variants that were difficult to characterize by standard short-read sequencing. Overall, these results demonstrate TaLon-SeqMD as an approach to provide rapid phased-variant calling to provide the molecular basis of Mendelian diseases.
尽管测序技术取得了进步，但许多孟德尔疾病患者仍无法进行分子诊断。目前的短读长临床测序方法无法提供染色体相位信息或表观遗传信息，而无需进一步的样本处理，但这种处理通常不会进行，可能导致患者的分子诊断不完整。能够从单个测序运行中提供定相的遗传和表观遗传信息，将提高孟德尔疾病的诊断率。在这里，我们描述了使用实时自适应测序方法对孟德尔疾病基因进行靶向长读测序（TaLon-SeqMD）。生物信息学靶向优化使人类基因组中多个致病区域的选择性富集成为可能。在单个测序运行中，可以实现单倍型解析的变异调用和同时解决表观遗传碱基修饰。在针对 373 个遗传性视网膜疾病（IRD）基因进行先前遗传测试的 18 名个体的队列中验证了 TaLon-SeqMD 方法，在每种情况下均实现了完整的分子诊断。然后将该方法应用于 2 例具有不确定测试结果的 IRD 病例，发现了难以通过标准短读长测序进行表征的非编码和结构变异。总体而言，这些结果表明 TaLon-SeqMD 是一种提供快速定相变异调用的方法，可提供孟德尔疾病的分子基础。
