Next-generation sequencing of custom amplicons to improve coverage of HaloPlex multigene panels
Next-generation sequencing of custom amplicons to improve coverage of HaloPlex multigene panels
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DOI：
10.2144/000114217
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发表时间：
2014-10-01
期刊：
影响因子：
2.7
通讯作者：
Kumanovics, Attila
中科院分区：
文献类型：
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作者：
Coonrod, Emily M.;Durtschi, Jacob D.;Kumanovics, Attila
用于遗传临床诊断的下一代多基因测序（NGS）要求100%覆盖所有目标基因。在遗传诊断实验室，在收集和分析NGS数据后，通常用Sanger测序来填补覆盖范围的空白。使用基于杂交的定制捕获HaloPlex方法制备的文库覆盖率接近98%，并且由于用于片段化的限制性内切酶位点的位置以及设计和实际文库插入大小的差异，在覆盖率上存在差距。我们描述了一种方法来提高HaloPlex文库的覆盖范围，通过生成一组扩增子，这些扩增子跨越已知的低覆盖区域，这些扩增子被合并，按样本索引，并与HaloPlex文库一起测序。该方法减少了所需的NGS后Sanger测序反应的数量，并且在需要完整基因覆盖时补充了任何NGS文库制备方法。
Next-generation sequencing (NGS) of multigene panels performed for genetic clinical diagnostics requires 100% coverage of all targeted genes. In the genetic diagnostics laboratory, coverage gaps are typically filled with Sanger sequencing after NGS data are collected and analyzed. Libraries prepared using the hybridization-based custom capture HaloPlex method are covered at similar to 98% and include gaps in coverage because of the location of the restriction enzyme sites used for fragmentation and differences in the designed and actual library insert size. We describe a method for improving the coverage of HaloPlex libraries by generating a set of amplicons spanning known low-coverage regions that are pooled, indexed by sample, and sequenced together with the HaloPlex libraries. This approach reduces the number of post-NGS Sanger sequencing reactions required and complements any NGS library preparation method when complete gene coverage is necessary.