title: HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing.
abstract: Super high-resolution single molecule sequence-based typing (SS-SBT) is a human leukocyte antigen (HLA) DNA typing method to the field 4 level of allelic resolution (formerly known as eight-digit typing) to efficiently detect new and null alleles without phase ambiguity by combination of long ranged polymerase chain reaction (PCR) amplification and next-generation sequencing (NGS) technologies. We previously reported the development and application of the SS-SBT method for the eight classical HLA loci, A, B, C, DRB1, DQA1, DQB1, DPA1 and DPB1. In this article, we describe the development of the SS-SBT method for three DRB1 linked loci, DRB3, DRB4 and DRB5 (DRB3/4/5) and characterization of DRB1-DRB3/4/5 haplotype structures to the field 4 level. Locus specific PCR primers for DRB3/4/5 were designed to amplify the gene regions from intron 1 to exon 6 [3' untranslated region (3'UTR)]. In total 20 DRB1 and 13 DRB3/4/5 allele sequences were determined by the SS-SBT to the field 4 level without phase ambiguity using 19 DR51, DR52 and DR53 positive genomic DNA samples obtained from Japanese. Moreover, 18 DRB1-DRB3/4/5 haplotypes were estimated to the field 4 level by the SS-SBT method in contrast to 10 haplotypes estimated by conventional methods to the field 1 level (formerly known as two digit typing). Therefore, DRB1-DRB3/4/5 haplotyping by SS-SBT is expected to provide informative data for improved HLA matching in medical research, transplantation procedures, HLA-related disease studies and human population diversity studies.
authors.affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ozaki
authors.forename: S
authors.initials: S
authors.lastname: Suzuki
authors.forename: A
authors.initials: A
authors.lastname: Shigenari
authors.forename: Y
authors.initials: Y
authors.lastname: Okudaira
authors.forename: E
authors.initials: E
authors.lastname: Kikkawa
authors.forename: A
authors.initials: A
authors.lastname: Oka
authors.forename: M
authors.initials: M
authors.lastname: Ota
authors.forename: S
authors.initials: S
authors.lastname: Mitsunaga
authors.forename: J K
authors.initials: JK
authors.lastname: Kulski
authors.forename: H
authors.initials: H
authors.lastname: Inoko
authors.forename: T
authors.initials: T
authors.lastname: Shiina
mesh_terms: D000483:Alleles; D017931:DNA Primers / Q000235:genetics; D005838:Genotype; D059811:HLA-DRB1 Chains / Q000235:genetics*; D059826:HLA-DRB3 Chains / Q000235:genetics*; D059845:HLA-DRB4 Chains / Q000235:genetics*; D059847:HLA-DRB5 Chains / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006650:Histocompatibility Testing / Q000379:methods* / Q000639:trends; D006801:Humans; D016133:Polymerase Chain Reaction; D014181:Transplantation Immunology
chemical_list: D017931:DNA Primers; D059811:HLA-DRB1 Chains; D059826:HLA-DRB3 Chains; D059845:HLA-DRB4 Chains; D059847:HLA-DRB5 Chains
keywords: DNA typing; human leukocyte antigen; next-generation sequencing; polymerase chain reaction; super high resolution single molecule - sequence-based typing