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

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