Resolving RH and GYP hybrid alleles while accessing the entire blood group genome with Nanopore adaptive sampling

Adaptive sampling (AS), a computational enrichment method developed for Oxford Nanopore Technologies sequencing platforms, offers a promising advance in molecular blood group diagnostics. By leveraging long-read sequencing, AS has the potential to accurately resolve complex structural variants in the RH and MNS blood group systems, while characterizing the entire blood group genome. In this study, we evaluated the performance of AS using five samples with known or suspected complex variants in the RH and MNS systems, unresolved by standard methods in immunohematology. Samples were sequenced on a PromethION P2 Solo with up to two samples per flowcell, generating 37.0-52.4 Gb of data with mean on-target coverages of 18.9-53.4x, allowing reliable variant detection. Hybrid alleles were characterized using a de novo assembly approach, whereas variants in non-recombinant regions were analyzed using both a custom in-house and the EPI2ME reference-based workflow. Considering solely exonic variation, 10-15% of detected alleles contained novel nonsynonymous single nucleotide variants (SNVs) or unreported SNV combinations. All suspected hybrid alleles were successfully assembled and identified as GYP*401.02, RHD*03N.01, and RHD*01EL.44, representing the first fully characterized haplotypes for these variants publicly available. Overall, AS showed significant potential for advancing blood group genomics by enabling high-resolution, full-gene analysis. Its ability to support high-throughput donor genotyping and precise patient-donor matching may reduce the risk of alloimmunization and delayed hemolytic transfusion reactions, particularly in chronically transfused patients. These findings highlight AS as a powerful tool for both research and clinical applications in transfusion medicine.