title: 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.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, 84108, Salt Lake City, UT.
authors.forename: Emily M
authors.initials: EM
authors.lastname: Coonrod
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, 84108, Salt Lake City, UT.
authors.forename: Jacob D
authors.initials: JD
authors.lastname: Durtschi
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, 84108, Salt Lake City, UT.
authors.forename: Chad
authors.initials: C
authors.lastname: VanSant Webb
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, 84108, Salt Lake City, UT; Department of Pathology, University of Utah School of Medicine, 84112, Salt Lake City, UT.
authors.forename: Karl V
authors.initials: KV
authors.lastname: Voelkerding
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, 84108, Salt Lake City, UT; Department of Pathology, University of Utah School of Medicine, 84112, Salt Lake City, UT.
authors.forename: Attila
authors.initials: A
authors.lastname: Kumánovics
mesh_terms: D015723:Gene Library; D005796:Genes; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*
keywords: HaloPlex; custom amplicons; multigene panels; next-generation sequencing; read coverage