title: HAPCAD: An open-source tool to detect PCR crossovers in next-generation sequencing generated HLA data.
abstract: Next-generation sequencing (NGS) based HLA genotyping can generate PCR artifacts corresponding to IMGT/HLA Database alleles, for which multiple examples have been observed, including sequence corresponding to the HLA-DRB1(∗)03:42 allele. Repeat genotyping of 131 samples, previously genotyped as DRB1(∗)03:01 homozygotes using probe-based methods, resulted in the heterozygous call DRB1(∗)03:01+DRB1(∗)03:42. The apparent rare DRB1(∗)03:42 allele is hypothesized to be a "hybrid amplicon" generated by PCR crossover, a process in which a partial PCR product denatures from its template, anneals to a different allele template, and extends to completion. Unlike most PCR crossover products, "hybrid amplicons" always corresponds to an IMGT/HLA Database allele, necessitating a case-by-case analysis of whether its occurrence reflects the actual allele or is simply the result of PCR crossover. The Hybrid Amplicon/PCR Crossover Artifact Detector (HAPCAD) program mimics jumping PCR in silico and flags allele sequences that may also be generated as hybrid amplicon.
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA; San Francisco State University, Department of Biology, 1600 Holloway Avenue, San Francisco, CA 94132, USA. Electronic address: shana.mcdevitt@gmail.com.
authors.forename: Shana L
authors.initials: SL
authors.lastname: McDevitt
authors.affiliation: Department of Molecular & Cell Biology, University of California, 142 LSA #3200, Berkeley, CA 94720, USA. Electronic address: jessenbredeson@berkeley.edu.
authors.forename: Jessen V
authors.initials: JV
authors.lastname: Bredeson
authors.affiliation: San Francisco State University, Department of Biology, 1600 Holloway Avenue, San Francisco, CA 94132, USA. Electronic address: scottwroy@gmail.com.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Roy
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA. Electronic address: jlane@chori.org.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Lane
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA. Electronic address: jnoble@chori.org.
authors.forename: Janelle A
authors.initials: JA
authors.lastname: Noble
mesh_terms: D016477:Artifacts; D019295:Computational Biology / Q000379:methods*; D030541:Databases, Genetic; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction; D012984:Software; D064878:Web Browser
chemical_list: D006680:HLA Antigens
keywords: Human Leukocyte Antigen; IMGT/HLA Database; Next-generation sequencing; Open-source tools; PCR crossovers