Department of Molecular Medicine and Surgery, Center for Molecular Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden.
Department of Clinical Genetics and Genomics, Karolinska University Hospital, 171 76 Stockholm, Sweden.
Genome Res. 2024 Nov 20;34(11):1774-1784. doi: 10.1101/gr.279510.124.
Clinical genetic laboratories often require a comprehensive analysis of chromosomal rearrangements/structural variants (SVs), from large events like translocations and inversions to supernumerary ring/marker chromosomes and small deletions or duplications. Understanding the complexity of these events and their clinical consequences requires pinpointing breakpoint junctions and resolving the derivative chromosome structure. This task often surpasses the capabilities of short-read sequencing technologies. In contrast, long-read sequencing techniques present a compelling alternative for clinical diagnostics. Here, Genomic Medicine Sweden-Rare Diseases has explored the utility of HiFi Revio long-read genome sequencing (lrGS) for digital karyotyping of SVs nationwide. The 16 samples from 13 families were collected from all Swedish healthcare regions. Prior investigations had identified 16 SVs, ranging from simple to complex rearrangements, including inversions, translocations, and copy number variants. We have established a national pipeline and a shared variant database for variant calling and filtering. Using lrGS, 14 of the 16 known SVs are detected. Of these, 13 are mapped at nucleotide resolution, and one complex rearrangement is only visible by read depth. Two Chromosome 21 rearrangements, one mosaic, remain undetected. Average read lengths are 8.3-18.8 kb with coverage exceeding 20× for all samples. De novo assembly results in a limited number of phased contigs per individual (N50 6-86 Mb), enabling direct characterization of the chromosomal rearrangements. In a national pilot study, we demonstrate the utility of HiFi Revio lrGS for analyzing chromosomal rearrangements. Based on our results, we propose a 5-year plan to expand lrGS use for rare disease diagnostics in Sweden.
临床遗传实验室通常需要对染色体重排/结构变异(SVs)进行全面分析,包括易位、倒位等大事件,以及额外的环状/标记染色体和小的缺失或重复。要了解这些事件的复杂性及其临床后果,需要确定断点连接,并解析衍生染色体结构。这项任务通常超出了短读测序技术的能力范围。相比之下,长读测序技术为临床诊断提供了一个引人注目的替代方案。在这里,瑞典罕见病基因组医学中心(Genomic Medicine Sweden-Rare Diseases)探索了 HiFi Revio 长读长基因组测序(lrGS)在全国范围内用于 SV 数字核型分析的应用。从瑞典所有医疗保健地区收集了来自 13 个家庭的 16 个样本。先前的研究已经确定了 16 个 SV,范围从简单到复杂的重排,包括倒位、易位和拷贝数变异。我们已经建立了一个全国性的管道和一个共享的变异数据库,用于变异调用和过滤。使用 lrGS,可检测到 16 个已知 SV 中的 14 个。其中 13 个在核苷酸分辨率下进行映射,一个复杂的重排只能通过读深度可见。两个 21 号染色体重排,一个是镶嵌型,仍然未被检测到。平均读长为 8.3-18.8kb,所有样本的覆盖率均超过 20×。从头组装的结果是每个个体的相位数目有限(N50 为 6-86Mb),能够直接对染色体重排进行特征描述。在全国性的试点研究中,我们展示了 HiFi Revio lrGS 用于分析染色体重排的实用性。根据我们的结果,我们提出了一个为期 5 年的计划,以扩大 lrGS 在瑞典罕见病诊断中的应用。
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