HLA sequencing
The human leukocyte antigen (HLA) region, also referred to as the major histocompatibility complex (MHC), spans over 4 Mb on chromosome 6 and plays a central role in normal immune functions, autoimmune diseases, and transplantation. Studying the HLA locus is essential to uncovering the mechanisms behind these processes; however, the HLA region presents many difficulties owing to its complexity and polymorphic nature. The ability to span large portions of the HLA region, if not the entire locus, with long nanopore reads, offers a promising solution.
Importance of the HLA system
Transplantation is the best treatment option for many types of end-stage diseases, improving both the quality and length of life. Underpinning the success of organ or bone marrow transplantation is HLA typing, which enables HLA matching of donors and recipients. Insufficient matching between donor and recipient antigens can result in fatal complications such as organ rejection or graft-versus-host disease (GvHD).
The HLA region is a highly polymorphic region located on chromosome 6. Typically, the epitopes with high antigenicity are coded for in exons 2 (HLA class I and II) and 3 (HLA class I) of the HLA genes— these are the protein binding elements of the major histocompatibility complex (MHC) (Figure 1). HLA alleles are defined by the SNP and indel combinations within single phased sequences, and specific nomenclature is used to define alleles. Analysing these variants forms the foundations of high-resolution typing and enhances the chances of successful transplantation.
Figure 1. MHC class II molecule: The α1 and β1 subunits of the protein binding domain are encoded for by exon 2 of their respective genes.
Figure 2: The large number of SNPs seen here when read mapping three human reference samples to the GRChH38 reference genome highlights the polymorphic nature of the HLA genes, with HLA-A shown as an example. Sequenced from 3 reference samples on a single MinION Flow Cell.
Resolving the HLA region
In recent years, next-generation sequencing (NGS), mainly based on short-read sequencing technologies, has superseded traditional HLA typing methods, offering improved throughput and resolution, plus reduced turnaround times. However, such methods succumb to the inherent limitations of short reads, which often align ambiguously to HLA alleles and make phasing of distant variants challenging.
The long sequencing reads that can be generated on Oxford Nanopore devices have been shown to overcome the associated issues of short-read sequencing and increase the accuracy of HLA typing. Long nanopore reads, capable of spanning the entire HLA region, transforms variant detection by readily linking together adjacent variants, enabling unambiguous phasing of SNVs to a respective haplotype. An efficient method for phased determination of HLA alleles will likely aid in the discovery of additional markers of importance that will improve transplantation success. Furthermore, using long nanopore reads, variants can be called within regulatory regions, and these have the potential to influence gene expression.
Demonstrating potential for rapid and accurate HLA typing with real-time nanopore sequencing
High-resolution, phased HLA typing data is necessary to ensure epitope matching between donor and recipient for solid organ transplantation. While short-read platforms can phase fragments of 400–900 bp, they are unable to sequence fragments spanning kilobases in single reads. Mosbruger et al. performed targeted sequencing of HLA class I and II loci on 120 samples. Twenty-four samples could be sequenced in multiplex on a single MinION Flow Cell in less than 24 hours, while single samples could be typed in less than six hours with Flongle; this compares to a 2–3 day turnaround time for short-read platforms. The authors found that the nanopore sequencing-based HLA typing workflow was 100% accurate at genotyping HLA-A, HLA-B, HLA-C, DPA1, DPB1, DQA1, DRB3 and DRB5.
Nanopore technology offers ' ... a significant advancement over current next-generation sequencing platforms as a single solution for all HLA genotyping needs'.
Mosbruger et al. 2020. Human Immunology.Nanopore sequencing ' ... allows us also to do the typing to the same level as we could with next generation sequencing, but we could do it faster, our sample prep was easier and it was much more cost efficient'.
Steven Verbruggen, OHMX.bioCharacterising the HLA region with PCR-free targeted nanopore sequencing
The HLA region is highly variable; accurate, phased HLA genotyping is needed to ensure the success of organ transplantation. Short-read methods for HLA typing suffer from PCR bias and phasing is difficult with short reads. These problems are overcome with PCR-free long nanopore sequencing reads. At London Calling 2022, Steven Verbruggen (OHMX.bio, Ghent) discussed his work using PCR-free targeted nanopore sequencing for HLA typing. Using adaptive sampling, which negates the need for any extra lab-based steps as enrichment is performed in real-time during sequencing, Steven achieved 10-20x enrichment of the HLA region. When using the Cas9 Sequencing Kit, up to 30-40x enrichment was achieved; enough signal for high-resolution HLA typing. Steven concluded that HLA typing with nanopore sequencing is cost efficient, easy, and provides detailed results faster than any alternative technologies.
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