RESUMEN
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.
Asunto(s)
Técnicas de Genotipaje/métodos , Antígenos HLA/genética , Secuenciación de Nanoporos/métodos , Alelos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Prueba de Histocompatibilidad/métodos , Humanos , Análisis de Secuencia de ADN/métodos , Donantes de TejidosRESUMEN
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 serologic 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 2-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 the cases. Several pre- and posttransplant 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 the cases required HR-2F typing by Sanger sequencing, as supported by other legacy methods, to properly address posttransplant anti-HLA antibody issues.
