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Combining one‐step Sanger sequencing with phasing probe hybridization for HLA class I typing yields rapid, G‐group resolution predicting 99% of unique full length protein sequences
Author(s) -
Tu Bin,
Masaberg Carly,
Hou Lihua,
Behm Daniel,
Brescia Peter,
Cha Nuri,
Kariyawasam Kanthi,
Lee Jar How,
g Thoa,
Sells John,
Tausch Paul,
Yang Ruyan,
Ng Jennifer,
Hurley Carolyn Katovich
Publication year - 2017
Publication title -
hla
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.347
H-Index - 99
eISSN - 2059-2310
pISSN - 2059-2302
DOI - 10.1111/tan.12951
Subject(s) - sanger sequencing , human leukocyte antigen , typing , biology , genetics , dna sequencing , genotype , allele , multilocus sequence typing , computational biology , dna , antigen , gene
Background Sanger‐based DNA sequencing of exons 2+3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. This study was undertaken to reduce the time and effort required to produce a single high resolution HLA genotype. Materials and methods Samples were typed in parallel by Sanger sequencing and oligonucleotide probe hybridization. This workflow, together with optimization of analysis software, was tested and refined during the typing of over 42,000 volunteers for an unrelated hematopoietic progenitor cell donor registry. Next generation DNA sequencing (NGS) was applied to over 1000 of these samples to identify the alleles present within the G group designations. Results Single genotypes at G level resolution were obtained for over 95% of the loci without additional assays. The vast majority of alleles identified (>99%) were the primary allele giving the G groups their name. Only 0.7% of the alleles identified encoded protein variants that were not detected by a focus on the antigen recognition domain (ARD)‐encoding exons. Conclusion Our combined method routinely provides biologically relevant typing resolution at the level of the ARD. It can be applied to both single samples or to large volume typing supporting either bone marrow or solid organ transplantation using technologies currently available in many HLA laboratories.