Abstract
BackgroundThe 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.ResultsA 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.ConclusionsOur protocol generated phased-defined sequences of the entire HLA genes, resulting in high resolution HLA typing and new allele detection.
Highlights
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
Strong associations between carbamazepineinduced Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) and HLA-B*15:02 [6,7], abacavir-induced liver injury and HLA-B*57:01 [8,9,10,11], and allopurinol-induced SJS or TEN and HLA-B*58:01 [12] have been reported in various populations
PCR primers were designed to anneal where known polymorphic sites were not observed according to the dbSNP build 135 database, and to amplify regions spanning the promoter to 3′UTR of the HLA genes (Additional file 1: Table S1)
Summary
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. Accurate, and cost-effective method for generating phase-defined complete sequencing of HLA genes by using indexed multiplex generation sequencing. The completion of a continuous 3.6 Mb of HLA genomic sequence together with annotation of 224 genes, was first reported by The MHC Sequencing Consortium in 1999 [2]. For better understanding of disease causality and drug hypersensitivity, phase-defined complete HLA gene sequencing is required. Complete HLA gene sequences are essential to minimize risk of graft versus host disease in hematopoietic transplantation because unknown determinants could be located around HLA genes. SBT or Sanger sequencing simultaneously sequences two chromosomes, thereby, phasing of the highly polymorphic HLA genes is very difficult per se. Allele determination is generally based on sequence alignment to the IMGT/HLA database where there is an inherent limitation
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