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Phase-defined complete sequencing of the HLA genes by next-generation sequencing.

ABSTRACT: BACKGROUND: The human leukocyte antigen (HLA) region, the 3.8-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, mostly autoimmune diseases. Due to the complex nature of HLA genes, there are difficulties in elucidating complete HLA gene sequences especially HLA gene haplotype structures by the conventional sequencing method. We propose a novel, accurate, and cost-effective method for generating phase-defined complete sequencing of HLA genes by using indexed multiplex next generation sequencing. RESULTS: A total of 33 HLA homozygous samples, 11 HLA heterozygous samples, and 3 parents-child families were subjected to phase-defined HLA gene sequencing. We applied long-range PCR to amplify six HLA genes (HLA-A, -C, -B, DRB1, -DQB1, and -DPB1) followed by transposase-based library construction and multiplex sequencing with the MiSeq sequencer. Paired-end reads (2?×?250 bp) derived from the sequencer were aligned to the six HLA gene segments of UCSC hg19 allowing at most 80 bases mismatch. For HLA homozygous samples, the six amplicons of an individual were pooled and simultaneously sequenced and mapped as an individual-tagging method. The paired-end reads were aligned to corresponding genes of UCSC hg19 and unambiguous, continuous sequences were obtained. For HLA heterozygous samples, each amplicon was separately sequenced and mapped as a gene-tagging method. After alignments, we detected informative paired-end reads harboring SNVs on both forward and reverse reads that are used to separate two chromosomes and to generate two phase-defined sequences in an individual. Consequently, we were able to determine the phase-defined HLA gene sequences from promoter to 3'-UTR and assign up to 8-digit HLA allele numbers, regardless of whether the alleles are rare or novel. Parent-child trio-based sequencing validated our sequencing and phasing methods. CONCLUSIONS: Our protocol generated phased-defined sequences of the entire HLA genes, resulting in high resolution HLA typing and new allele detection.

SUBMITTER: Hosomichi K

PROVIDER: S-EPMC3671147 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Phase-defined complete sequencing of the HLA genes by next-generation sequencing.

Hosomichi Kazuyoshi K Jinam Timothy A TA Mitsunaga Shigeki S Nakaoka Hirofumi H Inoue Ituro I

BMC genomics 20130528

<h4>Background</h4>The human leukocyte antigen (HLA) region, the 3.8-Mb segment of the human genome at 6p21, has been associated with more than 100 different diseases, mostly autoimmune diseases. Due to the complex nature of HLA genes, there are difficulties in elucidating complete HLA gene sequences especially HLA gene haplotype structures by the conventional sequencing method. We propose a novel, accurate, and cost-effective method for generating phase-defined complete sequencing of HLA genes ...[more]

PMID: 23714642

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Project description:Genotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA.Each HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database.The genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology.The performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.

Project description:Unraveling of the HLA-related immunogenetic basis of several immune disorders is complex due to the extensive HLA polymorphism and strong linkage-disequilibrium between HLA loci. A lack of in phase sequence information, a relative deficiency of high resolution genotyping including non-coding regions and ambiguous haplotype assignment make it difficult to compare findings across association studies and to attribute a causal role to specific HLA alleles/haplotypes in disease susceptibility and modification of disease phenotypes. Earlier, historical antibody and DNA-based methods of HLA typing, primarily of low resolution at antigen/alellic group levels, yielded "indicative" findings which were partially improved by high-resolution DNA-based typing. Only recently, next-generation sequencing (NGS) approaches based on deep-sequencing of the complete HLA genes combined with bioinformatics tools began to provide the access to complete information at an allelic level. Analyzing HLA with NGS approaches, therefore, promises to provide further insight in the etiopathogenesis of several immune disorders in which HLA associations have been implicated. These range from coeliac disease and rheumatological conditions to even more complex disorders, such as type-1 diabetes, systemic lupus erythematosus and sarcoidosis. A systemic disease of unknown etiology, sarcoidosis has previously been associated with numerous HLA variants and also other gene polymorphisms, often in linkage with the HLA region. To date, the biological significance of these associations has only partially been defined. Therefore, more precise assignments of HLA alleles/haplotypes using NGS approaches could help to elucidate the exact role of HLA variation in the multifaceted etiopathogenesis of sarcoidosis, including epigenetic mechanisms. NGS-based HLA analyses may be also relevant for defining variable clinical phenotypes and for predicting the disease course or the response to current/plausible novel therapies.

Dataset Information

Phase-defined complete sequencing of the HLA genes by next-generation sequencing.

Publications

Phase-defined complete sequencing of the HLA genes by next-generation sequencing.

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