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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >High-quality DNA sequence capture of 524 disease candidate genes
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High-quality DNA sequence capture of 524 disease candidate genes

机译:524个疾病候选基因的高质量DNA序列捕获

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摘要

The accurate and complete selection of candidate genomic regions from a DNA sample before sequencing is critical in molecular diagnostics. Several recently developed technologies await substantial improvements in performance, cost, and multiplex sample processing. Here we present the utility of long padlock probes (LPPs) for targeted exon capture followed by array-based sequencing. We found that on average 92% of 5,471 exons from 524 nuclear-encoded mitochondrial genes were successfully amplified from genomic DNA from 63 individuals. Only 144 exons did not amplify in any sample due to high GC content. One LPP was sufficient to capture sequences from < 100-500 bp in length and only a single-tube capture reaction and one microarray was required per sample. Our approach was highly reproducible and quick (<8 h) and detected DNA variants at high accuracy (false discovery rate 1%, false negative rate 3%) on the basis of known sample SNPs and Sanger sequence verification. In a patient with clinical and biochemical presentation of ornithine transcarbamy-lase (OTC) deficiency, we identified copy-number differences in the OTC gene at exon-level resolution. This shows the ability of LPPs to accurately preserve a sample's genome information and provides a cost-effective strategy to identify both single nucleo-tide changes and structural variants in targeted resequencing.
机译:测序前从DNA样品中准确,完整地选择候选基因组区域对于分子诊断至关重要。几种最新开发的技术正在等待性能,成本和多重样品处理的实质性改进。在这里,我们介绍了长挂锁探针(LPP)用于靶向外显子捕获,然后进行基于阵列的测序的实用程序。我们发现,从63个个体的基因组DNA中成功扩增了524个核编码线粒体基因的5,471个外显子中的平均92%。由于高GC含量,任何样品中只有144个外显子没有扩增。一个LPP足以捕获长度小于100-500 bp的序列,每个样品仅需一个单管捕获反应和一个微阵列即可。我们的方法具有很高的重现性和快速性(<8小时),并且基于已知的样品SNP和Sanger序列验证,可以高精度(错误发现率1%,错误阴性率3%)检测到DNA变异。在临床和生化方面存在鸟氨酸转氨酶(OTC)缺乏症的患者中,我们在外显子水平分辨率下确定了OTC基因的拷贝数差异。这显示了LPP准确保存样品的基因组信息的能力,并提供了一种经济有效的策略来识别靶向重测序中的单个核苷酸变化和结构变异。

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