title: Identification and characterization of novel HLA alleles: Utility of next-generation sequencing methods.
abstract: The HLA genes are the most polymorphic of the human genome, and novel HLA alleles are continuously identified, often by clinical Sanger sequencing-based typing (SBT) assays. Introduction of next-generation sequencing (NGS) technologies for clinical HLA typing may significantly improve this process. Here we compare four cases of novel HLA alleles identified and characterized by both SBT and NGS. The tested NGS system sequenced broader regions of the HLA loci, and identified novel polymorphisms undetected by SBT. Subsequent characterization of the novel alleles in isolation of coencoded alleles by SBT required custom-designed primers, while the NGS system was able to sequence both alleles in phase. However, the tested assay was unable to amplify buccal cell DNA for subsequent NGS sequencing, presumably due to the lower quality of these samples. While NGS assays will undoubtedly increase novel allele identification, more stringent DNA sample requirements may be necessary for this new technology.
authors.affiliation: Department of Pathology, University of Chicago Medicine, Chicago, IL, United States.
authors.forename: Nicholas K
authors.initials: NK
authors.lastname: Brown
authors.affiliation: Department of Pathology, University of Chicago Medicine, Chicago, IL, United States.
authors.forename: Taba
authors.initials: T
authors.lastname: Kheradmand
authors.affiliation: Department of Pathology, University of Chicago Medicine, Chicago, IL, United States.
authors.forename: Jinguo
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Pathology, University of Chicago Medicine, Chicago, IL, United States. Electronic address: smarino@bsd.uchicago.edu.
authors.forename: Susana R
authors.initials: SR
authors.lastname: Marino
mesh_terms: D000483:Alleles; D003062:Codon; D005091:Exons; D006680:HLA Antigens / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006650:Histocompatibility Testing; D006801:Humans; D017422:Sequence Analysis, DNA
chemical_list: D003062:Codon; D006680:HLA Antigens
keywords: Allele-specific primers; NGS; Next-generation sequencing; Novel HLA alleles; SBT; Sanger sequencing