Complete sequences of six Major Histocompatibility Complex haplotypes, including all the major MHC class II structures

Abstract

Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina and ultra-long Nanopore sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (B38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.32% – 0.69% in a 1000 Genomes Project read re-mapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.

Human leukocyte antigen super-locus: nexus of genomic supergenes, SNPs, indels, transcripts, and haplotypes

“…Initially, only two haplotypes were resolved completely at the base pair level (cell-lines PGF and COX); whereas the other six haplotypes were completed only at 51% (cell-line APD) to 93% (cell-line QBL) of the MHC genomic region. Seven of the SCMHP cell-lines were resequenced again as part of 95 near-complete haplotypes, using short-range and long-range NGS 11,49 . Overall, Norman et al provided 137 genotyped loci for most of the 95 celllines that they sequenced 11 .…”

“…The SNP count was <0.001 SNP/kb between the same HLA-A haplotypes in C and D. Spikes and peaks of SNPs above 100 SNP/kb were due mostly to nucleotide misalignments because of poor sequence assembly, structural variations, gaps, inversions or long runs of unspecified nucleotides. See recent SNP plots by Houwaart et al 49 for additional comparisons between MHC haplotypes. expression and disease, D'Antonio et al showed that eQTL analyses of HLA haplotypes provided substantially greater statistical power than only using single variants 59 .…”

HLA/MHC and KIR characterization in humans and non‐human primates using Oxford Nanopore Technologies and Pacific Biosciences sequencing platforms

“…Genome assemblies generated by such a hybrid approach using Nanopore and Illumina sequencing, for example, resolved and phased HLA haplotypes with a 99% accuracy at a 3-field resolution. 99,107 Another hybrid approach using PacBio and Hi-C sequencing, which is a high-throughput chromosome conformation capture technique, resolved HLA and KIR haplotypes with a 98%-99% accuracy in comparison to reference sequences. 108 Combining WGS data obtained from both a PacBio and an ONT platform resolved HLA regions with 100% accuracy, whereas another research group using this hybrid sequencing approach defined HLA and KIR haplotypes that contained only minor errors in repeat-rich segments.…”

“…Therefore, WGS on a PacBio or ONT platform is often combined with a short‐read method to ensure sufficient coverage that improves the accuracy. Genome assemblies generated by such a hybrid approach using Nanopore and Illumina sequencing, for example, resolved and phased HLA haplotypes with a 99% accuracy at a 3‐field resolution 99,107 . Another hybrid approach using PacBio and Hi‐C sequencing, which is a high‐throughput chromosome conformation capture technique, resolved HLA and KIR haplotypes with a 98%–99% accuracy in comparison to reference sequences 108 .…”

A pan-MHC reference graph with 246 fully contiguous phased sequences

“…Fully phased MHC sequences are invaluable resources because they can serve as references for imputation using short reads. Very few of these sequences exist today, to our knowledge, and they are mostly derived from individuals of European ancestry 32 , so are blinded to the great diversity of HLA alleles and haplotypes in non-European populations 25,[34][35][36][37] .…”