Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.
Hum Mutat. 2012 Apr;33(4):614-26. doi: 10.1002/humu.22032. Epub 2012 Feb 28.
In this study, we assess exome sequencing (ES) as a diagnostic alternative for genetically heterogeneous disorders. Because ES readily identified a previously reported homozygous mutation in the CAPN3 gene for an individual with an undiagnosed limb girdle muscular dystrophy, we evaluated ES as a generalizable clinical diagnostic tool by assessing the targeting efficiency and sequencing coverage of 88 genes associated with muscle disease (MD) and spastic paraplegia (SPG). We used three exome-capture kits on 125 individuals. Exons constituting each gene were defined using the UCSC and CCDS databases. The three exome-capture kits targeted 47-92% of bases within the UCSC-defined exons and 97-99% of bases within the CCDS-defined exons. An average of 61.2-99.5% and 19.1-99.5% of targeted bases per gene were sequenced to 20X coverage within the CCDS-defined MD and SPG coding exons, respectively. Greater than 95-99% of targeted known mutation positions were sequenced to ≥1X coverage and 55-87% to ≥20X coverage in every exome. We conclude, therefore, that ES is a rapid and efficient first-tier method to screen for mutations, particularly within the CCDS annotated exons, although its application requires disclosure of the extent of coverage for each targeted gene and supplementation with second-tier Sanger sequencing for full coverage.
在这项研究中，我们评估了外显子组测序（ES）作为遗传异质性疾病的诊断替代方法。由于 ES 很容易识别出一个未确诊的肢带型肌营养不良症个体中 CAPN3 基因的先前报道的纯合突变，因此我们通过评估与肌肉疾病（MD）和痉挛性截瘫（SPG）相关的 88 个基因的靶向效率和测序覆盖度，将 ES 评估为一种可推广的临床诊断工具。我们在 125 个人中使用了三种外显子组捕获试剂盒。使用 UCSC 和 CCDS 数据库定义了构成每个基因的外显子。三种外显子组捕获试剂盒靶向 UCSC 定义的外显子内的 47-92%的碱基和 CCDS 定义的外显子内的 97-99%的碱基。在 CCDS 定义的 MD 和 SPG 编码外显子中，每个基因的平均靶向碱基分别以 61.2-99.5%和 19.1-99.5%的比例以 20X 覆盖率进行测序。在每个外显子中，大于 95-99%的靶向已知突变位置以≥1X 覆盖率测序，55-87%以≥20X 覆盖率测序。因此，我们得出结论，ES 是一种快速有效的一线筛查突变的方法，特别是在 CCDS 注释的外显子中，尽管其应用需要披露每个靶向基因的覆盖程度，并补充二级 Sanger 测序以获得全面覆盖。
