Mosbruger Timothy L, Dinou Amalia, Duke Jamie L, Ferriola Deborah, Li Yang, Hayeck Tristan J, Monos Dimitri S
Immunogenetics Laboratory, Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States.
Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States.
Nucleic Acids Res. 2025 May 22;53(10). doi: 10.1093/nar/gkaf441.
Recently long-read sequencing technologies and bioinformatics have enabled the construction of haplotype-resolved genome assemblies. Here, we present the complete and accurate de novo characterization of two challenging genomic regions, the major histocompatibility complex (MHC) and Killer-cell immunoglobulin-like receptors (KIRs), in phased haplotypic form, using the Oxford Nanopore Technology (ONT) Adaptive Sampling sequencing, and a newly developed bioinformatics pipeline. These critical regions for our immune response have been notoriously difficult to characterize due to their sequence variability and structural complexity. The key features of our approach are (i) focused sequencing of specific regions, (ii) exclusive use of ONT, and (iii) a unique phasing methodology that integrates sequencing reads, methylation signals, and a reference panel. Ten samples with known MHC and KIR haplotypes were sequenced and assembled, demonstrating the potential of our approach. We achieved efficient target enrichment resulting in 100% coverage and accuracy ranging from 99.95% to 99.99% across the MHC and KIR. Its simplicity, reproducibility, and affordability distinguish this method as a unique and effective approach for the targeted haplotypic characterization of the MHC and KIR without trios and possibly other specific genomic regions. These efforts will in turn facilitate future studies that further advance the functional deconvolution of our genome.
最近，长读长测序技术和生物信息学使得构建单倍型解析的基因组组装成为可能。在此，我们展示了使用牛津纳米孔技术（ONT）自适应采样测序和新开发的生物信息学流程，以分阶段单倍型形式对两个具有挑战性的基因组区域——主要组织相容性复合体（MHC）和杀伤细胞免疫球蛋白样受体（KIR）进行完整且准确的从头表征。由于其序列变异性和结构复杂性，这些对我们免疫反应至关重要的区域一直以来都极难表征。我们方法的关键特征包括：（i）对特定区域进行靶向测序；（ii）仅使用ONT；（iii）一种独特的定相方法，该方法整合了测序读数、甲基化信号和一个参考面板。对十个已知MHC和KIR单倍型的样本进行了测序和组装，证明了我们方法的潜力。我们实现了高效的目标富集，在MHC和KIR区域实现了100%的覆盖，准确率在99.95%至99.99%之间。该方法的简单性、可重复性和可承受性使其成为一种独特且有效的方法，可用于在无三联体的情况下对MHC和KIR以及可能的其他特定基因组区域进行靶向单倍型表征。这些努力反过来将促进未来的研究，进一步推动我们基因组功能解卷积的进展。
