Assessment of HaloPlex Amplification for Sequence Capture and Massively Parallel Sequencing of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Genes
Assessment of HaloPlex Amplification for Sequence Capture and Massively Parallel Sequencing of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Genes
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DOI：
10.1016/j.jmoldx.2014.09.006
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发表时间：
2015-01-01
影响因子：
4.1
通讯作者：
Jonasson, Jon
中科院分区：
文献类型：
--
作者：
Green, Anna;Green, Henrik;Jonasson, Jon
致心律失常性右室心肌病（ARVC）的遗传基础是复杂的。编码心脏桥粒组分的基因突变与ARVC有因果关系。下一代测序允许同时对多个基因进行并行测序和重复/缺失分析，适用于筛查具有异质遗传背景的疾病中的突变。我们设计并验证了一个下一代的测序测试面板ARVC使用卤代。我们使用SureDesign制备了一个Haloadenovirus富集系统，用于使用MiSeq仪器对来自ARVC患者的DES、DSC 2、DSG 2、DSP、JUP、PKP 2、RYR 2、TGFB 3、TMEM 43和TIN进行测序。通过与桑格（作为金标准）和DSC 2、DSG 2、DSP、JUP和PKP 2的TruSeq定制扩增子测序进行比较，确定性能特征。所有样品在卤代磷酸酶捕获后成功测序，>99%的靶向核苷酸被> 20 x覆盖。这些序列具有高质量，尽管检测到由于推定的上下文特异性测序错误导致的位于DSP基因外显子1中的基序而导致的一个问题区域。通过桑格测序发现的突变也使用Haloclonal技术发现。根据生物信息学管道，灵敏度从99.3%变化到100%，特异性从99.90/0变化到100%。通过桑格和HaloGlycoprotein测序发现的三个变体位置由于覆盖丢失而被TruSeq Custom Amplicon遗漏。
The genetic basis of arrhythmogenic right ventricular cardiomyopathy (ARVC) is complex. Mutations in genes encoding components of the cardiac desmosomes have been implicated as being causally related to ARVC. Next-generation sequencing allows parallel sequencing and duplication/deletion analysis of many genes simultaneously, which is appropriate for screening of mutations in disorders with heterogeneous genetic backgrounds. We designed and validated a next-generation sequencing test panel for ARVC using HaloPlex. We used SureDesign to prepare a HaloPlex enrichment system for sequencing of DES, DSC2, DSG2, DSP, JUP, PKP2, RYR2, TGFB3, TMEM43, and TIN from patients with ARVC using a MiSeq instrument. Performance characteristics were determined by comparison with Sanger, as the gold standard, and TruSeq Custom Amplicon sequencing of DSC2, DSG2, DSP, JUP, and PKP2. All the samples were successfully sequenced after HaloPlex capture, with >99% of targeted nucleotides covered by >20x. The sequences were of high quality, although one problematic area due to a presumptive context-specific sequencing error causing motif Located in exon 1 of the DSP gene was detected. The mutations found by Sanger sequencing were also found using the HaloPlex technique. Depending on the bioinformatics pipeline, sensitivity varied from 99.3% to 100%, and specificity varied from 99.90/0 to 100%. Three variant positions found by Sanger and HaloPlex sequencing were missed by TruSeq Custom Amplicon owing to Loss of coverage.