HLA Typing for the Next Generation

Mayor, Neema P.; Robinson, James T.; McWhinnie, Alasdair; Ranade, Swati; Eng, Kevin; Midwinter, W; Bultitude, Will P.; Chin, Chen-Shan; Bowman, Brett; Marks, Patrick; Braund, Henny; Madrigal, J. Alejandro; Latham, Katy; Marsh, Steven G.E.

doi:10.1371/journal.pone.0127153

Cited by 127 publications

(123 citation statements)

References 28 publications

(29 reference statements)

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“…Briefly, PCR amplifications of HLA‐A, ‐B, ‐C, ‐DRB1, ‐DQB1 and ‐DPB1 were performed using DNA barcoded primers and protocols designed in‐house and described previously . The regions amplified for each of the HLA genes are shown in Table .…”

Section: Methodsmentioning

confidence: 99%

Single molecule real‐time DNA sequencing of HLA genes at ultra‐high resolution from 126 International HLA and Immunogenetics Workshop cell lines

Turner

Hayhurst

Hayward

et al. 2017

HLA

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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.

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Section: Methodsmentioning

confidence: 99%

Single molecule real‐time DNA sequencing of HLA genes at ultra‐high resolution from 126 International HLA and Immunogenetics Workshop cell lines

Turner

Hayhurst

Hayward

et al. 2017

HLA

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“…Full-length HLA gene sequences with high resolution are required to establish the Indian HLA database of ethnic Indian population. The PacBio sequencing is a single molecule sequencing platform which provides long, full-length and haplotype sequence information for HLA genes/ alleles [13]. We identified three novel HLA alleles from PacBio data.…”

Section: Discussionmentioning

confidence: 99%

“…As the HLA genes (A, B, C, DRB1 and DQB1) are more than 5 Kb, the 454 or other short read sequencing was not able to resolve haplotypes of donors. To overcome the phasing limitations across distant SNPs especially those found in class II genes [12], third generation sequencing technology has been utilised where full-length HLA genesare amplified using long range PCR and sequenced using single molecule real-time (SMRT) PacBio sequencing [13]. Pac-Bio sequencing of HLA genes provides high-resolution (6 to 8 digit) HLA allelic information for multiple HLA genes (6 genes) with phased SNPs [13].…”

Section: Introductionmentioning

confidence: 99%

Comparative Analyses of Low, Medium and High-Resolution HLA Typing Technologies for Human Populations

Gowda¹,

Ambardar²,

Dighe³

et al. 2016

J Clin Cell Immunol

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Human Leukocyte Antigen (HLA) encoding genes are part of the major histocompatibility complex (MHC) on human chromosome 6. This region is one of the most polymorphic regions in the human genome. Prior knowledge of HLA allelic polymorphisms is clinically important for matching donor and recipient during organ/tissue transplantation. HLA allelic information is also useful in predicting immune responses to various infectious diseases, genetic disorders and autoimmune conditions. India harbors over a billion people and its population is untapped for HLA allelic diversity. In this study, we explored and compared three HLA typing methods for South Indian population, using Sequence-Specific Primers (SSP), NGS (Roche/454) and single-molecule sequencing (PacBio RS II) platforms. Over 1020 DNA samples were typed at low resolution using SSP method to determine the major HLA alleles within the South Indian population. These studies were followed up with medium resolution HLA typing of 80 samples based on exonic sequences on the Roche/454 sequencing system and high-resolution (6-8 digit) typing of 8 samples for HLA alleles of class I genes (HLA-A, B and C) and class II genes (HLA-DRB1 and DQB1) using PacBio RS II platform. The long reads delivered by SMRT technology, covered the full-length class I and class II genes/alleles in contiguous reads including untranslated regions, exons and introns, which provided phased SNP information. We have identified three novel alleles from PacBio data that were verified by Roche 454 sequencing. This is the first case study of HLA typing using second and third generation NGS technologies for an Indian population. The PacBio platform is a promising platform for large-scale HLA typing for establishing an HLA database for the untapped ethnic populations of India.

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“…Historically, full‐gene sequencing of HLA genes has been difficult and expensive . Modern NGS and single‐molecule sequencing technologies, such as MinION, allow laboratories to more easily sequence a full‐length gene, rather than just exon regions .…”

Section: Introductionmentioning

confidence: 99%

Saddlebags: A software interface for submitting full‐length HLA allele sequences to the EMBL‐ENA nucleotide database

Matern

Groeneweg

Voorter

et al. 2017

HLA

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Submission of full-length HLA allele sequences presents a unique challenge, both for high-throughput sequencing laboratories and smaller diagnostic laboratories. HLA's extensive polymorphism means that accurate representation and annotation of allele sequence is of critical importance, and curators of nucleotide databases must establish submission formats to ensure high-quality data and prevent ambiguities. The IPD-IMGT/HLA database is established as the standard repository for HLA sequences, and it is a major goal of the 17th International HLA and Immunogenetics Workshop to fill the IPD-IMGT/HLA database with full-length HLA sequences. The process of preparing sequence annotation and metadata is cumbersome and error prone, and it is desirable to create a straightforward and concise method of preparing sequence submissions. We introduce Saddlebags, a software tool for rapid generation of HLA (novel) full-length allele sequence submissions. HLA allele sequences are submitted first to EMBL European Nucleotide Archive (EMBL-ENA), and metadata is gathered for subsequent preparation of an IPD-IMGT/HLA formatted submission. Combining these steps into a pipeline reduces effort and minimizes errors for submitting laboratories. This software has been used by Maastricht University Medical Center Transplantation Immunology Laboratory to submit 79 novel alleles to EMBL-ENA, and the tool is freely available for the HLA community.

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HLA Typing for the Next Generation

Cited by 127 publications

References 28 publications

Single molecule real‐time DNA sequencing of HLA genes at ultra‐high resolution from 126 International HLA and Immunogenetics Workshop cell lines

Single molecule real‐time DNA sequencing of HLA genes at ultra‐high resolution from 126 International HLA and Immunogenetics Workshop cell lines

Comparative Analyses of Low, Medium and High-Resolution HLA Typing Technologies for Human Populations

Saddlebags: A software interface for submitting full‐length HLA allele sequences to the EMBL‐ENA nucleotide database

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