Science and Technology Department, BGI-Shenzhen, Shenzhen, China ; Department of Biology, University of Copenhagen, Copenhagen, Denmark.
PLoS One. 2013 Jul 24;8(7):e69388. doi: 10.1371/journal.pone.0069388. Print 2013.
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.
主要组织相容性复合体 (MHC) 是人类基因组中最具变异性和基因密度的区域之一。大多数 MHC 及其相关区域的研究都集中在次要变体和 HLA 分型上，其中许多已被证明与人类疾病易感性和代谢途径有关。然而，MHC 区域的变体检测和诊断 HLA 分型仍然缺乏一种连贯、标准化、具有成本效益且高覆盖率的临床质量和可靠性协议。在本文中，我们提出了一种使用高通量、高覆盖率的靶向区域测序来准确检测人类 MHC 区域中的次要变体和 HLA 类型的方法。设计了一个探针集来模板化 8 个注释的人类 MHC 单倍型，并包含扩展 MHC 区域的 500 万个碱基对 (Mb)。我们在三个遗传多样化的人类样本上部署了我们的探针，用于探针集评估，测序数据表明，∼97%的 MHC 区域和超过 99%的 MHC 区域中的基因都具有足够的深度和良好的均匀性。通过这种捕获测序调用的 98%的基因型与已建立的 HapMap 基因型一致。我们还同时开发了一种从这种靶向捕获测序数据中调用 IMGT/HLA 数据库中引用的任何 HLA 类型的一步式流水线，当以 4 个数字分辨率部署时，其分型准确率超过 96%。这种用于 MHC 区域中变体检测和 HLA 分型的经济高效且高度准确的方法可能会进一步深入研究免疫介导的疾病，并可能在移植医学研究中找到临床应用。这个一步式流水线是为科学界的一般评估和使用而发布的。
