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.
- Resolve and phase the entire HLA locus with long and ultra-long nanopore reads
- Scale to your requirements — from portable sequencing devices to modular, on-demand benchtop sequencers
- Achieve rapid sample-to-answer times with real-time sequencing
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 single nucleotide polymorphism (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.
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In recent years, next-generation sequencing (NGS), mainly based on short-read sequencing technologies, has superseded traditional HLA typing methods, offering improved throughput and resolution, plus reduced turnaround times.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.
Ultra-rapid, high-resolution HLA class I typing for pharmacogenetic testing
HLA genes have been shown to be involved in hypersensitivity to many drugs, with specific HLA class I alleles being most frequently implicated. In Thailand, HLA genotyping is routinely performed prior to the administration of prescription drugs; however, existing PCR-based genotyping assays often lack resolution and accuracy. To overcome these challenges, Anukul et al. utilised nanopore sequencing to deliver cost-effective and highly accurate (three- to four-field), same-day HLA typing of twenty clinical research samples.
Characterising 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 Cas9 sequencing, 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.
Flexible, population-scale sequencing using up to 48 independent, high-capacity flow cells — complete genomic and transcriptomic characterisation of large sample numbers.
Combining up to 24 independently addressable, high-capacity flow cells with powerful, integrated compute, PromethION 24 delivers flexible, on-demand access to terabases of sequencing data — ideal for cost-effective, high-throughput sequencing.
Flexible, high-yield nanopore sequencing for every lab. The PromethION 2 devices are designed to be compact and accessible, utilising 2 PromethION Flow Cells that can generate hundreds of gigabases of data each.
From genome assembly to gene expression, run multiple experiments on-demand using five independent MinION or Flongle Flow Cells.
Integrated sequencing and analysis in a powerful handheld device — suitable for small animal genomes, targeted sequencing, and gene expression studies.
Adapting MinION and GridION for smaller, routine tests and analyses. Low plex targeted sequencing, RNA isoform analysis, and quality control applications.
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