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Eight novel HLA class I alleles have been identified using full gene sequencing., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Two novel alleles, HLA-G*01:04:09 and HLA-DPB1*04:01:01:136, were identified in a single healthy individual., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DPB1 is the classical HLA class II genes with the least recorded variation on the IPD-IMGT/HLA Database, suggesting the full extent of its diversity is perhaps yet to be characterized. Here, a full-gene typing strategy was employed to genotype a UK cohort of 1470 HCT recipients (n = 744) and donors (n = 726). In total, 2940 full-length HLA-DPB1 sequences were generated, comprising 193 distinct alleles. Of these, 107 sequences contained novel variation, totaling 49 unique intronic HLA-DPB1 alleles, and one coding variant (HLA-DPB1*1188:01). Full-gene sequencing resulted in zygosity changes for 129 individuals by identifying two distinct intronic variants of the same coding allele. We verified the existence of nine unconfirmed alleles and extended the sequence of two existing alleles on the IPD-IMGT/HLA Database., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Two novel non-classical HLA class I alleles have been characterized, HLA-F*01:16 and -F*01:17., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Until recently the number of alleles of the nonclassical HLA class I gene HLA-E documented in the IPD-IMGT/HLA Database was small and as a result, the gene was often not considered to be notably polymorphic. Here, we describe our work in identifying and submitting 86 novel HLA-E alleles after full-gene single-molecule real-time (SMRT) DNA sequencing of 6227 DNA samples. These samples were comprised of 2468 patients undergoing hematopoietic cell transplantation and 3759 unrelated potential donors. A total of 111 unique HLA-E alleles were detected in this cohort. The majority of novel alleles (79.1%) contained polymorphisms in intronic regions, highlighting the significant undiscovered variation present in the noncoding regions of the HLA-E gene., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pacific Biosciences SMRT sequencing used to identify the novel allele, HLA-A*02:01:02:206., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-E*01:03:02:25 sequenced from blood and buccal samples of an ALL patient., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Two novel HLA class II alleles were characterized, HLA-DRB1*03:01:01:05 and HLA-DPB1*04:02:01:21., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

As the primary genetic determinant of immune recognition of self and non-self, the hyperpolymorphic HLA genes play key roles in disease association and transplantation. The large, variably sized HLA class II genes have historically been less well characterized than the shorter HLA class I genes. Here, we have used Pacific Biosciences Single Molecule Real-Time (SMRT®) DNA sequencing to perform four-field resolution HLA typing of HLA-DRB1/3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 from a panel of 181 B-lymphoblastoid cell lines from the International HLA and Immunogenetics Workshops. By interrogating all exons, introns, and the untranslated regions of these important reference cells, we have improved their HLA typing resolution on the IPD-IMGT/HLA database. We observed widespread non-coding polymorphism, with over twice as many unique genomic sequences identified compared with coding sequences (CDS). We submitted 263 unique sequences to the IPD-IMGT/HLA Database, often from multiple cell lines, including 114 confirmations of existing alleles, of which 30 were also extensions to full-length genomic sequences where only CDS was available previously. A total of 149 novel alleles were identified, largely differing from their closest reference allele sequences by a single nucleotide polymorphism (SNP). However, some highly divergent alleles were deemed to be recombinants, only detectable by full-length sequencing with long, phased reads. The fourth-field variation we observed allowed fine mapping of linkage disequilibrium patterns and haplotypes to particular ancestries. This study has highlighted the under-appreciated non-coding diversity in HLA class II genes, with potential implications for population genetic and clinical studies., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A novel variant of HLA-C*03:04:01:47 was identified using Pacific Biosciences SMRT sequencing platform., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We have developed a genotyping assay that produces fully phased, unambiguous HLA-E genotyping using Pacific Biosciences' single molecule real-time DNA sequencing. In total 212 cell lines were genotyped, including the panel of 107 established at the 10th International Histocompatibility Workshop. Our results matched the previously known HLA-E genotype in 94 (44.3%) cell lines, in all cases either improving or equalling previous genotyping resolution. Three (1.4%) cells had discrepant HLA-E genotyping data and 115 (54.2%) had no previous HLA-E data. The HLA-E genotypes for four (1.9%) cell lines resulted in a change of zygosity by identifying two distinct haplotypes. We discovered eight novel HLA-E alleles, extended the known reference sequence of seven and confirmed the existence of a further 10., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The assignment of an HLA allele name to a sequence requires a comparison between the generated target sequence and a reference sequence on the IPD-IMGT/HLA database. Absence of a full-length reference sequence can result in the inability of HLA typing software to accurately compare and assign the sequence. We sequenced the most frequently seen HLA class I alleles on the Anthony Nolan register present in the database with only a partial genomic sequence, with the aim of increasing the number of complete reference sequences. We successfully extended 95 full-length HLA class I sequences and identified 13 novel variants. Increasing the number of full-length HLA class I reference sequences in the database has aided accuracy of HLA analysis tools for all histocompatibility and immunogenetics laboratories., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The novel allele, HLA-C*07:01:01:30 differs by one nucleotide substitution with HLA-C*07:01:01:01 at gDNA 627., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-DRB1*01:01:01:02 is the first reported intronic variant of DRB1*01:01:01, differing by a single nucleotide substitution., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pacific Biosciences' SMRT sequencing method was used to extend the sequence of HLA-A*02:13., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pacific Bioscience's SMRT sequencing was used to confirm and extend HLA-DPB1*64:01N and 701:01., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pacific Bioscience's SMRT DNA sequencing was used to identify two novel intronic variants of HLA-A*32:01:01:01., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The novel KIR2DL1*037 allele discovered and characterised by single molecule real-time (SMRT) DNA sequencing., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Full-length KIR2DL1 allele sequence extensions characterised by single molecule real-time (SMRT) DNA sequencing., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B-lymphoblastoid cell lines (B-LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B-LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing to HLA type 126 B-LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra-high resolution. Amplicon sequencing of full-length HLA class I genes (HLA-A, -B and -C) and partial length HLA class II genes (HLA-DRB1, -DQB1 and -DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD-IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four-field resolution typing of HLA-B and HLA-C. By improving and standardising the HLA typing of these B-LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)