Open AccessNext-generation sequencing of custom amplicons to improve coverage of HaloPlex multigene panels
Author(s) -
Emily Coonrod,
Jacob D. Durtschi,
Chad VanSant Webb,
Karl V. Voelkerding,
Attila Kumánovics
Publication year - 2014
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/000114217
Subject(s) - sanger sequencing , amplicon , insert (composites) , dna sequencing , computational biology , biology , amplicon sequencing , restriction enzyme , genetics , gene , polymerase chain reaction , engineering , mechanical engineering , 16s ribosomal rna
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.