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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.

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.

Aim This study examined the variation observed when typing exons 2 + 3 of HLA-DQB1 using reverse sequence specific oligonucleotides (rSSO) as compared to typing exon 2 alone in volunteers from an unrelated hematopoietic stem cell donor program. New alleles and those with incomplete sequences were characterized with next generation sequencing (NGS) to update the IPD-IMGT/HLA database. Methods Samples were typed for HLA-DQB1 using One Lambda’s SSO LabType with additional phasing probes for exon 2 ( ∼ 50,000 donors) or for exons 2 + 3 ( ∼ 15,000). Full-length HLA-DQB1 sequences (40) were obtained using an Illumina platform and in-house protocol. Results Overall, 38 incomplete DQB1 alleles were identified and sequenced with NGS, including 6 that could only be detected by rSSO with exon 3 probes. The majority of these alleles appear only 1–2 times in the sample set and were not listed as common or well-documented in Mack et al. 2013. The addition of exon 3 in rSSO typing complicated analysis since many alleles lack sequence data for exon 3, and the software assumes no probe binding if there is no known sequence. An example is DQB1*06:08 ( ∼ 10), which binds to one of the exon 3 probes and therefore is not assigned (i.e., false positive). Two alleles showed discrepant results between rSSO and NGS due to novel sequences. The full-length sequences obtained for all alleles sequenced demonstrate high conservation of the regions outside of exon 2. Conclusions There are advantages and disadvantages from screening exons 2 + 3 of HLA-DQB1. It elucidates greater variation not identified from screening exon 2 alone but has a more complicated analysis due to incomplete DNA sequences. Updating the IPD-IMGT/HLA database is critical for improved accuracy. C. Masaberg: 7. Other (Identify); Company/Organization; Intellectual property typing workflow, One Lambda licensing. C.K. Hurley: 7. Other (Identify); Company/Organization; Intellectual property typing workflow, One Lambda licensing.

Aim The aim of this study was to complete full gene sequences for previously reported HLA class I sequences for the IPD-IMGT/HLA database. Methods We identified 569 class I alleles in ∼ 99,000 individuals typed for a U.S. donor registry that had incomplete sequences in the IPD-IMGTHLA database. The alleles had been typed by a combined Sanger SBT-SSOP workflow focused on exons 2 + 3. Single locus alleles were amplified with primers annealing in the 5’ and 3’ UTR and the sequences of exons and introns determined with an Illumina Miseq using an in-house protocol. Results Most (423, 74%) of the 569 alleles were found in a single cell. The majority of the class I full length sequences were identical to the most similar HLA allele (e.g., C*01:70 differed by only a single exon 2 nucleotide when compared to C*01:02:01:01). Two novel HLA-A alleles with exon 2/3 variation were not detected by SBT/SSO due to inability to phase key polymorphic nucleotides [A*03:150, *26:01:01G vs. A*03:01:01:01 + A*26:149 (new); A*02:01:55 + *33:03:01G vs. A*02:01:01:01 + *33:03:33 (new)]. Overall, new alleles were found in 18 (3.2%) samples; most in the targeted incomplete allele. Most (14, 78%) showed variation in intron and UTR regions. Some of the incomplete alleles (49/569) could not be characterized because the allele combination did not allow phasing of the sequences (e.g., C*07:01:01:01 + *07:27:01 had no polymorphic nucleotides within a span of 2298 bp (introns 2–7)). Conclusions Class I gene sequences outside exons 2 + 3 are conserved. Genotypes with novel alleles can be missed by typing assays that do not provide comprehensive phasing. Increasing the knowledge of full sequences for all alleles will improve the accuracy of HLA typing. Download : Download high-res image (153KB) Download : Download full-size image J.S. Ng: 4. Scientific/Medical Advisor; Company/Organization; Georgetown University. C.K. Hurley: 4. Scientific/Medical Advisor; Company/Organization; Georgetown University.

Aim Long range next generation sequencing was used to characterize the HLA class I (HLA-A,B,C) and class II (DRB1, DRB3/4/5, DQB1) alleles of a hematopoietic stem cell registry population (N = 643) from The Netherlands (Matchis). Methods The in-house typing protocol included several new strategies to reduce errors, add automation, and balance the yield of all loci when 96 samples are sequenced in one 600 cycle paired-end Illumina MiSeq sequence run. Results Of the 22 new class I alleles, three contain a nonsynonymous substitution in an exon (1, 4 or 7). The remainder are single nucleotide intron variants. Thirty-two class I alleles are incomplete in the IMGT/HLA database. The majority of the alleles in the population are common; the three most common alleles at each locus are listed in the table. Alleles not previously listed as common or well-documented include mainly 4th field variations; exceptions included single copies of B*51:20, C*07:100, and DRB1*04:92. Alleles encoding nonsynonymous substitutions compared to the primary allele in the G group include, as examples, A*23:17 (1 vs. 20 A*23:01:01:01), B*44:27:01 (1 vs 83 B*44:02:01:01), C*07:06 (3 vs 190 C*07:01:01:01), DRB1*14:54:01 (44 vs 3 DRB1*14:01:01), DQB1*02:02:01 (84 vs 175 DQB1*02:01:01). Non-expressed alleles were not observed. DRB3/4/5 associations were identified. Conclusions Long-range sequencing of HLA alleles from world-wide populations will continue to build our understanding of HLA diversity and its impact on the immune response. Download : Download high-res image (231KB) Download : Download full-size image C.K. Hurley: 7. Other (Identify); Company/Organization; Intellectual property sequencing Thermo Fisher.

Aim The goal of this study was to update the 2012 common and well documented (CWD) allele list using data from 3940 class I and 4124 class II assignments obtained by next generation sequencing (NGS) of two registry (USA and Argentina) populations and 360 donor-recipient pairs from the CIBMTR repository. Methods DNA was prepared from blood, buccal swabs, or cell lines. HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs. The sequences of exons and introns were determined by next generation sequencing (NGS) with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Class II NGS sequencing focused on exons 2 and 3; however, some alleles had additional exons and introns characterized. Alleles that were on the well-documented list and alleles not considered CWD based on Mack et al. Tissue Antigens 2013 were identified. Results The majority of alleles detected had previously been designated as common. For example, from the Argentine registry samples, 92% of 2944 HLA-A alleles identified were common. In the total study population, 77 class I alleles previously designated as WD and 97 alleles not defined as C or WD were observed from 1 to >5 times. Thirty-seven of the 174 class I alleles occurred greater than 5 times, e.g., previously undefined A*02:17:02, B*44:02:01:03, and C*17:01:01:05. The majority (90%) of the 19 not CWD alleles were intron variants e.g., A*03:01:01:05, B*39:01:01:03, C*06:02:01:03. Forty-five class II alleles fell into the WD or not defined categories; 29% occurred >5 times. Class II alleles occurring >5 times included DRB1*04:06:02, DQB1*03:01:01:03 and DPB1*04:02:01:02. Conclusions The NGS data expand our knowledge of the frequency of specific full length HLA alleles. The majority of the uncommon alleles characterized in this study are synonymous variations of common alleles.

Aim The aim of this study was to use next generation DNA sequencing (NGS) to characterize full length HLA class I gene diversity in 1472 volunteers for the unrelated donor registry in Argentina. Methods DNA was prepared from blood. HLA-A, -B, -C alleles were amplified with primers annealing in the 5′ and 3′ UTRs. The sequences of exons and introns were determined by NGS with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Analysis used the IPD-IMGT/HLA database from October 2016. Results Volunteers were recruited from 20 of the 23 provinces of Argentina and from its federal capital. The majority (80%) came from the central region of the country. Of the 2944 alleles identified at each locus, the majority have been designated as common (e.g., 92% HLA-A alleles) (Mack et al. Tissue Antigens 2013) and approximately 1% were not found in the IPD-IMGT/HLA database. A total of 79 different allele sequences were identified at the HLA-A locus with similar numbers at HLA-B and -C. The majority of the HLA proteins can be distinguished by sequencing only the antigen recognition domain (ARD)-encoding exons. For example, 94% of the HLA-A alleles could have been detected solely based on the sequence of the ARD-encoding exons. The remaining alleles differed only outside of these exons by either silent variation occurring mainly in introns (6%) or by nonsynonymous variation ( Conclusions NGS has allowed us to evaluate the frequency and characteristics of class I DNA sequence variation outside of the ARD-encoding exons. The additional variation detected is predominantly in noncoding regions. While NGS offers a strategy to achieve single genotype resolution, the amount of additional information gained related to HLA matching for transplantation is minimal.

Aim The aim of this study was to characterize the complete class I gene sequences of new class I alleles found in a population study of 1472 volunteers from the Argentina unrelated donor registry. Methods HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs and the sequences of exons and introns determined by next generation sequencing (NGS) with an Illumina Miseq using an in–house protocol and Conexio Assign software analysis. Initially class I and II amplicons were combined for sequencing; single locus sequencing of a second PCR amplicon was used to confirm variation. Allele sequences were submitted to the IPD-IMGT/HLA database. Results 1% of the 2944 alleles identified at each class I locus were novel. Three novel alleles were found multiple times. The majority (92%) differed by only a single nucleotide substitution from the known allele. Variants in noncoding regions predominated (79%) (T1). None of the intron variants appeared to impact RNA splicing motifs. The distribution of novel substitutions among introns is listed in T2. Unique substitutions, not shared by any other known allele at the locus, were found in 71% HLA-A, 58% HLA-B and 67% HLA-C novel alleles. Conclusions Sixty-six novel class I alleles were identified. The majority of substitutions were found in introns and appear to be random rather than resulting from gene conversion events. Download : Download high-res image (295KB) Download : Download full-size image Download : Download high-res image (73KB) Download : Download full-size image

Aim This study used next generation sequencing (NGS) in 1472 healthy volunteers from an Argentinian registry to characterize class II gene diversity. Complications of using NGS for these regions arise from large introns, different length alleles, regions with few polymorphisms, and incomplete sequences in the IPD-IMGT/HLA database. Methods Amplicons were generated to include exons 2–6 of HLA-DQB1, and exons 2–3 of -DRB1 and -DPB1 using DNA extracted from blood. Samples were combined and sequenced on an Illumina MiSeq with an in-house library construction protocol. Conexio Assign was used to identify alleles based on the IPD-IMGT/HLA database from October 2016. Full length gene sequencing of select samples was performed with two overlapping amplicons for HLA-DPB1 and -DRB1. Results The majority of the alleles identified at each locus were common with ⩽1% of alleles per gene designated as well-documented or with frequency not defined. With only exons 2–3 sequenced, 6% of HLA-DRB1 assignments and 11% of -DPB1 were ambiguous. When additional exons were sequenced, most showed the common allele; however, DPB1∗104:01 was seen in 16% of samples typed as DPB1∗03:FNVX. For HLA-DRB1, preferential amplification due to differences in allele size and presence of pseudogenes led to certain alleles with low read depth and poor sequence quality, in particular DRB1∗04. Analysis of HLA-DQB1 introns proved difficult due to insertions and deletions that poorly aligned to the reference. Intron 2 of HLA-DPB1 is large and has few variations between certain combinations of alleles that were not phased in 14% of samples. These were not resolved with full length sequencing. Conclusions Although conditions still need to be optimized, NGS of HLA class II is a powerful tool in determining genetic diversity. HLA-DRB1 ambiguities would be reduced by 82% with the initial sequencing of exons 2–4; however, longer amplicons reduce read depth. Addition of primers to maximize amplification of low amplifying alleles may be necessary to increase read depth. Alternate methodology such as sequencing of single DNA molecules is necessary to solve the ambiguities for alleles that are not phased. These proposed changes will lead to more successful sequencing of HLA class II. Even so, these complications make NGS challenging when applied to high volume typing.