Abstract
HLA alleles are observed in specific haplotypes, due to linkage disequilibrium (LD) between particular alleles. Haplotype frequencies for alleles have been established for specific ethnic groups and racial categories from population studies. HLA typing using Next Generation Sequencing (NGS) platforms allows generating full-length gene sequences, obtaining four-field HLA types and detecting novel alleles. NGS technologies also permit high-throughput HLA typing for large numbers of samples in a cost effective manner. Aim To establish HLA haplotypes with four-field resolution from quartet (two children and two parents) families. Methods We selected quartet families from our clinical database. Extended family members, aunt, uncle, cousin and grandparents, were also included if available. HLA types generated using SBT, SSP and SSO were reviewed for selecting subjects. Two children were selected if they did not share two HLA haplotypes. DNA was processed using MIA FORA NGS kits, and sequenced on either MiSeq or NextSeq sequencer. HLA types were obtained using MIA FORA 2.0 software. HLA haplotypes were built by comparing children’s and parents’ HLA types. The HLA haplotypes were reported according to the order of genes along the chromosome to identify potential meiotic recombination, and were also compared to the previously established haplotypes. Results Of 100 selected families, we sequenced 50 families. We built 200 haplotypes consisting of four-field HLA types from these. We developed automated haplotype building software. It was feasible to resolve almost all ambiguities including phase ambiguities and to generate completely phased full-length gene sequences from the consensus sequences generated from these studies. Conclusions The haplotypes generated from quartet families will be important resources for hematopoietic stem cell donor match prediction and play a role as reference haplotypes for expanding a collection of HLA haplotypes.
Published Version
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