Project Scientist UCLA Immunogenetics Center, University of California Los Angeles Los Angeles, CA
Aim: High-resolution human leukocyte antigen (HLA) gene typing is critical for transplantation, disease association studies, and immunogenetics. Accurate genotyping of class I and II HLA loci is essential for matching in bone marrow and solid organ transplantation, directly influencing graft survival and outcomes. Next-generation sequencing (NGS) enabled allele-level typing. However, due to variability in polymorphism and structural complexity, especially in class II genes, pose challenges for short-read sequencing often results in ambiguous or incomplete allelic-resolution.
Methods: This study validated a full-gene HLA typing workflow using Oxford Nanopore Technologies (ONT) and the GenDx protocol. We sequenced 175 samples across 11 classical loci (HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1, -DPB1). Each flow cell was reused for more than four runs without loss in quality, efficiently processing 24 samples at a time, while maintaining high pore availability.
Results: The HLA typing protocol showed unambiguous assignment of HLA genotypes for all loci. The mean read lengths for Class I were 3.1 kb and Class II 4.7 kb. The average read depth was 1278, and the minimum depth was 50. Mapping efficiency ranged from 73.0% to 98.0%, with noise below 17.4%. The average delta signal-to-noise ratio was 20.8% and the minimal estimated second allele percentage was as following: HLA-A (41%), -B (44%), -C (46%), -DRB1 (29%), -DQA1 (43%), -DQB1 (37%), -DPA1 (46%), and -DPB1 (39%). Allele frequencies detected in this cohort were consistent with common, intermediate and well-documented (CIWD). In addition, all novel alleles previously identified by our current NGS test were accurately detected, highlighting the platform's ability to resolve low-frequency variants. Significantly, the platform improved resolution for complex loci such as DPB1.
Conclusion: This ONT-based strategy offers a rapid, scalable, and accurate solution for full-gene HLA genotyping. Its strengths in phasing and speed make it highly valuable for transplantation and immunogenetics.
