High-throughput, high-fidelity HLA genotyping with deep sequencing

Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94003, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2012; 109(22):8676-81. DOI: 10.1073/pnas.1206614109
Source: PubMed


Human leukocyte antigen (HLA) genes are the most polymorphic in the human genome. They play a pivotal role in the immune response and have been implicated in numerous human pathologies, especially autoimmunity and infectious diseases. Despite their importance, however, they are rarely characterized comprehensively because of the prohibitive cost of standard technologies and the technical challenges of accurately discriminating between these highly related genes and their many allelles. Here we demonstrate a high-resolution, and cost-effective methodology to type HLA genes by sequencing, which combines the advantage of long-range amplification, the power of high-throughput sequencing platforms, and a unique genotyping algorithm. We calibrated our method for HLA-A, -B, -C, and -DRB1 genes with both reference cell lines and clinical samples and identified several previously undescribed alleles with mismatches, insertions, and deletions. We have further demonstrated the utility of this method in a clinical setting by typing five clinical samples in an Illumina MiSeq instrument with a 5-d turnaround. Overall, this technology has the capacity to deliver low-cost, high-throughput, and accurate HLA typing by multiplexing thousands of samples in a single sequencing run, which will enable comprehensive disease-association studies with large cohorts. Furthermore, this approach can also be extended to include other polymorphic genes.

Download full-text


Available from: Marcelo Fernande, Apr 18, 2014
    • "In recent years, various next generation sequencing platforms, 51 based on massively parallel clonal sequencing, have been used to 52 develop high resolution and high throughput HLA typing systems 53 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]. In general, two basic strategies have been used: (1) an 54 amplicon sequencing approach focused on the highly polymorphic 55 regions (primarily exons) of HLA class I and class II genes [1] [2] [3] [4] [5] [6], or 56 (2) long-range PCR of full or partial length individual HLA gene loci, 57 followed by fragmentation, shot-gun sequencing and assembly [7– 58 11]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Compared to Sanger sequencing, next-generation sequencing offers advantages for high resolution HLA genotyping including increased throughput, lower cost, and reduced genotype ambiguity. Here we describe an enhancement of the Roche 454 GS GType HLA genotyping assay to provide very high resolution (VHR) typing, by the addition of 8 primer pairs to the original 14, to genotype 11 HLA loci. These additional amplicons help resolve common and well-documented alleles and exclude commonly found null alleles in genotype ambiguity strings. Simplification of workflow to reduce the initial preparation effort using early pooling of amplicons or the Fluidigm Access Array™ is also described. Performance of the VHR assay was evaluated on 28 well characterized cell lines using Conexio Assign MPS software which uses genomic, rather than cDNA, reference sequence. Concordance was 98.4%; 1.6% had no genotype assignment. Of concordant calls, 53% were unambiguous. To further assess the assay, 59 clinical samples were genotyped and results compared to unambiguous allele assignments obtained by prior sequence-based typing supplemented with SSO and/or SSP. Concordance was 98.7% with 58.2% as unambiguous calls; 1.3% could not be assigned. Our results show that the amplicon-based VHR assay is robust and can replace current Sanger methodology. Together with software enhancements, it has the potential to provide even higher resolution HLA typing. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · May 2015 · Human immunology
    • "As a first step toward the establishment of a general HLA typing method (HLATyphon), we began with a algorithm similar to that of Wang et al. [11] and further optimized it to deal with the comparatively short reads generated by the MiSeq and to remove intronic and intergenic reads. First, the Sequence Polymorphism (SP) Reference Panel was obtained from the International Histocompatibility Working Group [4] "
    [Show abstract] [Hide abstract]
    ABSTRACT: We report the development of a general methodology to genotype HLA class I and class II loci. A Whole Genome Amplification (WGA) step was used as a sample sparing methodology. HLA typing data could be obtained with as few as 300 cells, underlining the usefulness of the methodology for studies for which limited cells are available. The next generation sequencing platform was validated using a panel of cell lines from the International Histocompatibility Working Group (IHWG) for HLA-A, -B, and -C. Concordance with the known, previously determined HLA types was 99%. We next developed a panel of primers to allow HLA typing of alpha and beta chains of the HLA DQ and DP loci and the beta chain of the DRB1 locus. For the beta chain genes, we employed a novel strategy using primers in the intron regions surrounding exon 2, and the introns surrounding exons 3 through 4 (DRB1) or 5 (DQB1 and DPB1). Concordance with previously determined HLA Class II types was also 99%. To increase throughput and decrease cost, we developed strategies combining multiple loci from each donor. Multiplexing of 96 samples per run resulted in increases in throughput of approximately 8-fold. The pipeline developed for this analysis (HLATyphon) is available for download at Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · May 2015 · Human immunology
  • Source
    • "Peptide specific responses were expressed as spot-forming cells (SFCs)/10 6 peripheral-blood mononuclear cells (PBMCs). Donors were HLA typed at each class II locus to four-digit resolution by SSP or deep sequencing methods [11] "
    [Show abstract] [Hide abstract]
    ABSTRACT: Computational prediction of HLA class II restricted T cell epitopes has great significance in many immunological studies including vaccine discovery. With the development of novel bioinformatics approaches, prediction of HLA class II binding has improved significantly but a strategy to predict the most dominant HLA class II epitopes has not been defined. Using different sets of peptides from various allergen and bacterial antigens and HLA class II binding prediction tools from the IEDB, we have designed a strategy to predict the top epitopes from any antigen. We found that the top 21% of 15-mer peptides overlapping by 10 residues (based on the predicted binding to seven DRB1 and DRB3/4/5 alleles) capture 50% of the immune response. This corresponded to an IEDB consensus percentile rank of 19.82 which could be used as a universal prediction threshold.
    Preview · Article · Sep 2014
Show more