The comprehensive characterization of human leukocyte antigen (HLA) genomic sequences remains a challenging problem. Despite the significant advantages of next-generation sequencing (NGS) in the field of Immunogenetics, there has yet to be a single solution for unambiguous, accurate, simple, cost-effective, and timely genotyping necessary for all clinical applications. This report demonstrates the benefits of nanopore sequencing introduced by Oxford Nanopore Technologies (ONT) for HLA genotyping. Samples (n = 120) previously characterized at high-resolution three-field (HR-3F) for 11 loci were assessed using ONT sequencing paired to a single-plex PCR protocol (Holotype) and to two multiplex protocols OmniType (Omixon) and NGSgo®-MX6-1 (GenDx). The results demonstrate the potential of nanopore sequencing for delivering accurate HR-3F typing with a simple, rapid, and cost-effective protocol. The protocol is applicable to time-sensitive applications, such as deceased donor typings, enabling better assessments of compatibility and epitope analysis. The technology also allows significantly shorter turnaround time for multiple samples at a lower cost. Overall, the nanopore technology appears to offer a significant advancement over current next-generation sequencing platforms as a single solution for all HLA genotyping needs.

The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly. RSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3’-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %. RSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing templates amenable to DNA sequencing on recently developed platforms. Although our demonstration of the method does not utilize these DNA sequencing platforms directly, our results indicate that the capture of long DNA fragments produce superior coverage of the targeted region.

HLA typing in solid organ transplantation (SOT) is necessary for determining HLA matching status between donor‐recipient pairs and assessing patients’ anti‐HLA antibody profiles. Histocompatibility has traditionally been evaluated based on serologically‐defined HLA antigens. The evolution of HLA typing and antibody identification technologies, however, has revealed many limitations with using serological equivalents for assessing compatibility in SOT. The significant improvements to HLA typing introduced by next‐generation sequencing (NGS) require an assessment of the impact of this technology on SOT. We have assessed the role of high‐resolution two‐field HLA typing (HR‐2F) in SOT by retrospectively evaluating NGS‐typed pre‐ and post‐ SOT cases. HR‐2F typing was highly instructive or necessary in 41% (156/385) of cases. Several pre‐ and post‐transplant scenarios were identified as being better served by HR‐2F typing. Five different categories are presented with specific case examples. The experience of another center (Temple University Hospital) is also included, whereby 21% of cases required HR‐2F typing by Sanger sequencing, as supported by other legacy methods, to properly address post‐transplant anti‐HLA antibody issues.

We describe a novel functional role for the HLA-B locus, mediated by its intron-encoded miRNA, miR-6891-5p. We show that in vitro inhibition of miR-6891-5p impacts the expression of nearly 200 transcripts within the B-lymphoblastoid cell line (B-LCL) COX, affecting a large number of metabolic pathways, including various immune response networks. Amongst the top affected transcripts following miR-6891-5p inhibition are those encoding the heavy chain of IgA. We identified a conserved miR-6891-5p target site on the 3’UTR of both IGHA1 and IGHA2 transcripts, and demonstrated that this miRNA modulates the expression of IGHA1 and IGHA2. B-LCLs from IgA deficient patients expressed significantly elevated levels of miR-6891-5p as compared to unaffected family members. Upon inhibition of miR-6891-5p, IgA mRNA expression levels were increased and IgA secretion was restored in the B-LCL of an IgA deficient patient. These findings indicate that miR-6891-5p regulates IGHA1 and IGHA2 gene expression at the post-transcriptional level and suggest that increase in miR-6891-5p levels may contribute to the etiology of selective IgA deficiency.