High-Resolution HLA Class I Genotyping Using G-Seq Platform-Based Nanopore Long-Read Sequencing Integrated With the...

This study evaluated the performance and clinical applicability of a novel third-generation sequencing (TGS) strategy for high-resolution HLA class I genotyping, employing nanopore long-read sequencing combined with a dedicated analytical workflow, HLATyper. A total of 49 clinical samples were analysed in parallel using conventional PCR-based sequence-based typing (PCR-SBT) and a TGS-based approach on the G-seq500 platform with the in-house-developed HLATyper pipeline. Genotyping results for HLA-A, -B and -C loci were compared between the two methods with respect to concordance, typing resolution and the ability to resolve ambiguous allele assignments. Ambiguity rates between the two methods were evaluated using Fisher's exact test. Complete concordance was observed between PCR-SBT and the TGS method at the two-field level across all samples. Ambiguous allele assignments were detected in 63.27% (31/49) of cases using PCR-SBT, whereas the TGS strategy produced a single, unambiguous genotype for every sample (p < 0.0001). Furthermore, TGS enabled assignment of four-field typing through comprehensive sequence alignment and successfully resolved ambiguities and exonic coverage gaps inherent to short-read approaches. G-seq500 platform-based nanopore long-read sequencing combined with the HLATyper analytical pipeline enables high-resolution, unambiguous and comprehensive HLA class I genotyping. This integrated strategy represents a valuable complementary approach for clinical applications requiring precise HLA matching, such as transplantation and platelet transfusion support.