Development of a high-resolution NGS-based HLA-typing and analysis pipeline

Abstract

The human leukocyte antigen (HLA) complex contains the most polymorphic genes in the human genome. The classical HLA class I and II genes define the specificity of adaptive immune responses. Genetic variation at the HLA genes is associated with susceptibility to autoimmune and infectious diseases and plays a major role in transplantation medicine and immunology. Currently, the HLA genes are characterized using Sanger- or next-generation sequencing (NGS) of a limited amplicon repertoire or labeled oligonucleotides for allele-specific sequences. High-quality NGS-based methods are in proprietary use and not publicly available. Here, we introduce the first highly automated open-kit/open-source HLA-typing method for NGS. The method employs in-solution targeted capturing of the classical class I (HLA-A, HLA-B, HLA-C) and class II HLA genes (HLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, HLA-DPB1). The calling algorithm allows for highly confident allele-calling to three-field resolution (cDNA nucleotide variants). The method was validated on 357 commercially available DNA samples with known HLA alleles obtained by classical typing. Our results showed on average an accurate allele call rate of 0.99 in a fully automated manner, identifying also errors in the reference data. Finally, our method provides the flexibility to add further enrichment target regions.

Integrative omics for health and disease

“…However, mortality among transplant patients remains very high. A standard practice for matching donors with recipients involves human leukocyte antigen (HLA) typing, for which methods have been recently developed using high-throughput sequencing technologies 123,124 . However, it is becoming increasingly clear that non-HLA factors can considerably affect prognosis and development of graft-versus-host disease (GVHD), as HLA-matched sibling donor transplants convey a lower risk of GVHD than HLA-matched but unrelated donor transplants 125 , and common non-HLA polymorphisms have been associated with GVHD 126 .…”

High-Accuracy HLA Type Inference from Whole-Genome Sequencing Data Using Population Reference Graphs

“…The current gold standard for high resolution typing of HLA alleles, sequence-based typing (SBT), uses Sanger sequencing or targeted amplification of the HLA genes followed by next-generation sequencing. With the growth of high throughput genomic technologies, methods for inferring HLA genotype have been developed that use SNP genotyping [9–12] or next-generation sequencing [13–19]. These approaches, to date, are either limited to a subset of HLA loci, require targeted capture / amplification, or do not achieve the same degree of accuracy as SBT.…”

High resolution HLA haplotyping by imputation for a British population bioresource

“…These include high resolution HLA haplotype frequencies in US populations for the entire US donor registry [7] and large scale data for German donors [8], [9] while databases of allelic reference sequences and nomenclature are maintained by IPD-IMGT/HLA (http://www.ebi.ac.uk/imgt/hla) [10]. There are a range of methods for direct HLA typing including serological testing, use of sequence-specific amplification primers (SSP) or probes (SSO), Sanger sequencing and next generation sequencing based typing [11], [12]. Imputation of HLA alleles from SNP genotyping [13], [14], [15], [16], [17] provides a further complementary approach of significant interest given the low cost and broad availability of accurate high throughput genotyping through genome-wide association studies and other initiatives.…”