An Integrated Tool to Study MHC Region: Accurate SNV Detection and HLA Genes Typing in Human MHC Region Using Targeted High-Throughput Sequencing

Abstract

The major histocompatibility complex (MHC) is one of the most variable and gene-dense regions of the human genome. Most studies of the MHC, and associated regions, focus on minor variants and HLA typing, many of which have been demonstrated to be associated with human disease susceptibility and metabolic pathways. However, the detection of variants in the MHC region, and diagnostic HLA typing, still lacks a coherent, standardized, cost effective and high coverage protocol of clinical quality and reliability. In this paper, we presented such a method for the accurate detection of minor variants and HLA types in the human MHC region, using high-throughput, high-coverage sequencing of target regions. A probe set was designed to template upon the 8 annotated human MHC haplotypes, and to encompass the 5 megabases (Mb) of the extended MHC region. We deployed our probes upon three, genetically diverse human samples for probe set evaluation, and sequencing data show that ∼97% of the MHC region, and over 99% of the genes in MHC region, are covered with sufficient depth and good evenness. 98% of genotypes called by this capture sequencing prove consistent with established HapMap genotypes. We have concurrently developed a one-step pipeline for calling any HLA type referenced in the IMGT/HLA database from this target capture sequencing data, which shows over 96% typing accuracy when deployed at 4 digital resolution. This cost-effective and highly accurate approach for variant detection and HLA typing in the MHC region may lend further insight into immune-mediated diseases studies, and may find clinical utility in transplantation medicine research. This one-step pipeline is released for general evaluation and use by the scientific community.

The Role of MHC-E in T Cell Immunity Is Conserved among Humans, Rhesus Macaques, and Cynomolgus Macaques

“…Sequencing of Mamu-E genomic DNA to identify novel Mamu-E alleles was performed using NimbleGen HLA SeqCap reagents to capture genomic MHC sequences from 31 rhesus macaques as described by Cao and colleagues (47). The captured MHC products were subsequently sequenced with Illumina HiSeq following the manufacturer’s instructions.…”

A regulatory mutant on TRIM26 conferring the risk of nasopharyngeal carcinoma by inducing low immune response

“…This approach has proved to be a cost‐effective solution for studying MHC region,16 particularly facilitating the population‐based research. Consequently, in this study, we applied this approach to conduct a targeted MHC sequencing in 40 Cantonese NPC patients followed by a two‐stage population‐based replication to identify the causal MHC loci or genes contributing to NPC.…”

The novel HLA‐C*08:80 allele identified by full‐length sequencing of the HLA region

“…Here, we describe a novel allele, HLA‐C*08:80 , which was identified by analyzing the DNA sequencing data of a Han Chinese individual. This allele was identified by HLA typing using the Sanger sequence‐based typing (Sanger SBT) method, and was further confirmed by next‐generation sequencing.…”

“…The result of Utype3.0 did not match with any known HLA alleles, suggesting the possible existence of either a novel HLA‐C*08:01 ‐like or C*15:02 ‐like allele with a single nucleotide polymorphism (SNP) at position 142 nt G‐>T. In order to confirm the allele, the entire 5 Mb MHC region, was designed and captured by Nimblegen Human MHC design probers (Roche Nimblegen, Inc., Madison, Wisconsin). The amplified library was then loaded to Illumina HiSeq2000 sequencer (Illumina, Inc., San Diego, California), and sequencing reads were aligned to the reference hg19 by Burrows‐Wheeler Aligner (BWA, version 0.7.8‐r455, Wellcome Trust Sanger Institute, Cambridge, UK), then, SOAPHLA were further used for phasing. The result of haplotype analysis suggested the presence of a novel HLA‐C allele, identical C*08:01:01:01 except a single nucleotide substitution at position 142 of exon 2, with G substituted by T, resulting in a coding change alanine to serine (Figure ).…”