Next-generation sequencing of custom amplicons to improve coverage of HaloPlex multigene panels.

Abstract

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 ~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.