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 quantity of life. Underpinning the success of transplantation is HLA typing; insufficient matching between donor and recipient antigens can result in fatal complications such as organ rejection or graft-versus-host disease (GvHD). 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). Therefore, analysing variants within these genomic regions 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In recent years, next-generation sequencing (NGS), mainly based on short-read sequencing technologies, has superseded traditional HLA typing methods, offering improved throughput and resolution, plus reduced turnaround times.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 (Shafin et al.). 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 (Figure 2). 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.
Assembly and phasing of the 4-Mb HLA locus
‘Ultra-long reads enabled assembly and phasing of the 4-Mb major histocompatibility complex (MHC) locus in its entirety’
Using long and ultra-long reads generated on a MinION device, Jain and colleagues assembled the genome of the human GM12878 cell line. The HLA class I and much of the repetitive class II locus was assembled in a single contig. The team subsequently resolved the HLA locus from the diploid HG00733 genome that had been sequenced on a PromethION. Again, a single contig spanned the entire HLA region, and the haplotypes of the diploid genome could be phased.
Also based at the University of California Santa Cruz, Shafin et al. later benchmarked the performance of various sequencing technologies for their ability to call SNVs in highly repetitive and difficult to map regions. Their results showed that Oxford Nanopore outperformed short-read and long-read sequencing platforms at SNV calling in the HLA region.
Rapid high-resolution HLA typing of deceased donor organ
‘The ability to perform comprehensive, high-resolution HLA typing for deceased organ donor allocation prior to transplantation would have major clinical benefits, particularly for highly sensitised recipients’
The window for transplantation is very narrow in cases where the graft is from a deceased donor; in such instances, rapid typing of donors is critical. The importance of high-resolution HLA typing cannot be overstated; HLA epitopes located on the surface of the donor’s HLA molecules are key determinants for the success of solid organ transplants. However, the use of short-read sequencing platforms to perform high-resolution typing of donors is precluded due to the long turnaround times. To that end, De Santis et al. developed a rapid, high-resolution HLA typing method, using the Oxford Nanopore Flongle platform. Amplicons targeting the 11 HLA loci were sequenced, with two-field typing achieved in just four hours. Complete concordance at two-field resolution was demonstrated between the Flongle-based assay and the existing typing methodology.
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