HLA-DRB1, -DRB3, -DRB4 and -DRB5 genotyping at a super-high resolution level by long range PCR and high-throughput sequencing

Tissue Antigens. 2014 Jan;83(1):10-6. doi: 10.1111/tan.12258. Epub 2013 Nov 30.


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.

Keywords: DNA typing; human leukocyte antigen; next-generation sequencing; polymerase chain reaction; super high resolution single molecule - sequence-based typing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • DNA Primers / genetics
  • Genotype
  • HLA-DRB1 Chains / genetics*
  • HLA-DRB3 Chains / genetics*
  • HLA-DRB4 Chains / genetics*
  • HLA-DRB5 Chains / genetics*
  • High-Throughput Nucleotide Sequencing
  • Histocompatibility Testing / methods*
  • Histocompatibility Testing / trends
  • Humans
  • Polymerase Chain Reaction
  • Transplantation Immunology


  • DNA Primers
  • HLA-DRB1 Chains
  • HLA-DRB3 Chains
  • HLA-DRB4 Chains
  • HLA-DRB5 Chains