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Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, -DRB3/4/5, and -DQB1 assignments of 1009 unrelated volunteers for the unrelated donor registry in The Netherlands. The analysis characterizes all HLA exons and introns for class I alleles; at least exons 2 to 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 229 class I and 71 class II; 36 of these alleles are novel. The majority (approximately 98%) of the cumulative allele frequency at each locus is contributed by alleles that appear three or more times. Alleles encoding protein variation outside of the antigen recognition domains are 0.6% of the class I assignments and 5.3% of the class II assignments.

Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, and -DQB1 assignments of 1472 unrelated volunteers for the unrelated donor registry in Argentina. The analysis characterized all HLA exons and introns for class I alleles; at least exons 2, 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 330 class I and 98 class II. The majority (~98%) of the cumulative allele frequency at each locus is contributed by alleles that appear at a frequency of at least 1 in 1000. Fourteen (18.2%) of the 77 novel class I and II alleles carry nonsynonymous variation within their exons; 52 (75.4%) class I novel alleles carry only single, apparently random, nucleotide variation within their introns/untranslated regions. Alleles encoding protein variation not usually detected by typing focused only on the exons encoding the antigen recognition domain are 1.0% of the class I assignments and 7.3% of the class II assignments (predominantly DQB1*02:02:01, DQB1*03:19:01, and DRB1*14:54:01). Updates to the common and well documented list of alleles include 10 alleles previously thought to be uncommon but that are found at least 30 times. Five locus haplotypes estimated using the expectation-maximization algorithm as present 3 or more times total 187. While the known HLA diversity continues to increase, the conservation of known allele sequences is remarkable. Overall, the HLA diversity observed in the Argentinian population reflects its European and Native American ancestry.

Aim Sanger-based DNA sequencing of exons 2 + 3 of HLA class I alleles from a heterozygote frequently results in two or more alternative genotypes. In order to reduce the time and effort required to produce a single high resolution HLA genotype, samples were typed in parallel by Sanger sequencing and probe hybridization to obtain G level typing resolution. Methods The panel of probes used was designed to yield high to intermediate resolution typing and to provide the phasing necessary to resolve common alternative genotypes. Class I assignments from more than 30,000 samples from registry donors were typed using this strategy supported by an in-house data merger and management software. To monitor accuracy and resolution, 552 samples were retyped by a next generation full length gene sequencing (NGS) strategy. Results More than 95% of the class I assignments were at the level of a single G-level genotype compared to ∼ 26% for European Americans when probe data were not included. The work load was reduced by at least 70% by eliminating secondary sequencing assays and by dramatically reducing the time needed for data review since the combined method addresses the weaknesses of each single method. Typing accuracy was increased. 99% of Sanger + probe typing results were concordant with NGS; all 32 allele discrepancies were attributed to weaknesses in the NGS data. Furthermore, NGS also demonstrated that only 0.9% of the G level assignments resulted in different full length protein sequences (e.g., C∗07:01:01G yielded C∗07:18 with NGS). The combined method had a rapid turnaround time compared with NGS and previous methods applied alone. Conclusions Our combined method routinely provides biologically relevant typing resolution at the level of the antigen recognition domain. It can be applied to both single sample or to large volume typing supporting either bone marrow or solid organ transplantation using technology currently available in many HLA laboratories and is cost effective when compared with current NGS kits. B. Tu: Other (Identify); Company/Organization; Intellectual property. C. Masaberg: Other (Identify); Company/Organization; Intellectual property. J. Lee: Employee; Company/Organization; Thermo Fisher. D. Behm: Employee; Company/Organization; Data Blueprint. J. Sells: Employee; Company/Organization; Data Blueprint. P. Tausch: Employee; Company/Organization; Thermo Fisher. L. Hou: Other (Identify); Company/Organization; Intellectual property. J. Ng: Other (Identify); Company/Organization; Intellectual property. C.K. Hurley: Other (Identify); Company/Organization; Intellectual property.

HLA class I assignments were obtained at single genotype, G-level resolution from 98 855 volunteers for an unrelated donor registry in the United States. In spite of the diverse ancestry of the volunteers, over 99% of the assignments at each locus are common. Within this population, 52 novel alleles differing in exons 2 and 3 are identified and characterized. Previously reported alleles with incomplete sequences in the IPD-IMGT/HLA database (n = 519) were selected for full gene sequencing and, from this sampling, another 27 novel alleles are described.

Traditional DNA-based typing focuses primarily on interrogating the exons of human leukocyte antigen (HLA) genes that form the antigen recognition domain (ARD). The relevance of mismatching donor and recipient for HLA variation outside the ARD on hematopoietic stem cell transplantation (HSCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in 10 of 10 (HLA-A, -B, -C, -DRB1, -DQB1) matched unrelated donor transplant pairs (n = 360). Next-generation DNA sequencing was used to characterize both HLA exons and introns for HLA-A, -B, -C alleles; exons 2, 3 and the intervening intron for HLA-DRB1 and exons only for HLA-DQA1 and -DQB1. Over 97% of alleles at each locus were matched for their nucleotide sequence outside of the ARD exons. Of the 4320 allele comparisons overall, only 17 allele pairs were mismatched for non-ARD exons, 41 for noncoding regions and 9 for ARD exons. The observed variation between donor and recipient usually involved a single nucleotide difference (88% of mismatches); 88% of the non-ARD exon variants impacted the amino acid sequence. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D-R pairs will be difficult to assess in HSCT outcome studies because these mismatches do not occur very frequently.