Introduction: Xenotransplantation with genetically modified pig organs represents a promising approach to address the shortage of human donor organs. Xenocompatibility between recipient human leukocyte antigens (HLA) and donor swine leukocyte antigens (SLA) plays a key role in the immune response to xenografts. Accurate SLA genotyping in donor pigs is essential for assessing potential cross-reactivity with HLA antibodies and understanding its clinical relevance. This knowledge is vital for informing targeted genetic modifications to reduce immunogenicity, improve xenocompatibility, and support long-term graft survival. Here, we introduce a rapid and accurate method for high resolution SLA genotyping using third-generation long-read sequencing technology.
Methods: Sequencing was performed on the Oxford Nanopore Technologies MinION Mk1B platform. Data analysis and typing assignments were conducted using a research prototype of the GenDx NGSengine-Turbo software pre-configured with all officially designated SLA sequences and alleles from the IPD-MHC database v3.12.0.0.
Results: Locus-specific primers were designed to amplify all classical SLA genes (SLA-1, -2, -3, -DRA, -DRB1, -DQA, -DQB1). Whole-gene amplification was achieved for all loci except DRB1, where exon 1 was excluded due to the presence of an unusually long intron 1. A total of 65 samples, sourced from both outbred commercial farm pigs and inbred research miniature pigs, were included in the validation and optimization process. Typing results were cross-referenced with previously determined low-resolution genotypes, as well as high-resolution haplotypes and alleles inferred from published SLA nomenclature reports. The method demonstrated a high level of concordance and successfully identified novel alleles that had previously been missed by prior low-resolution typing approaches. The sample cohort encompassed 46 distinct SLA haplotypes (comprising 30 unique class I and 22 unique class II haplotypes) with over 130 alleles, representing over 30% of the 426 officially designated alleles across the 7 targeted SLA genes. Our protocol enables multiplex sequencing and analysis of up to 24 samples per run, with a typical turnaround time of under 12 hours.
Conclusion: In summary, we have developed a rapid, high-resolution SLA typing system leveraging third-generation long-read sequencing to address the growing demand for improved xenotransplantation strategies. By enabling accurate characterization of SLA alleles in donor pigs, this system enhances our understanding of xenocompatibility and facilitatea more precise matching between donor pigs and human recipients, ultimately promoting the success and long-term survival of xenografts. The ability to process multiple samples per run with a fast turnaround time underscores the system’s efficiency, practicality, and scalability for both research and clinical applications.