Proof-of-principle: nanopore adaptive sampling enables full blood group genome analysis and resolution of hybrid alleles

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 through a simple, fast and locus-adjustable protocol. As proof-of-principal, we evaluated the performance of AS using 5 samples with suspected complex variants in the RH and MNS systems, unresolved by standard immunohematological methods. Samples were sequenced on a PromethION P2 Solo with up to 2 samples per flowcell, generating 37.0 to 52.4 gigabases of data with mean on-target coverages of 18.9× to 53.4×, allowing reliable variant detection. Hybrid alleles were characterized using a de novo assembly approach, whereas variants in nonrecombinant regions were analyzed using both a custom in-house and the EPI2ME reference-based workflow. With reference to field-specific allele collections, 10% to 15% of detected alleles contained novel nonsynonymous single-nucleotide variants (SNVs) or unreported exonic SNV combinations. All suspected hybrid alleles were successfully assembled and identified as GYP∗401.02, RHD∗03N.01, and RHD∗01EL.44, representing, to our knowledge, 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 patients who receive chronic transfusions. These findings highlight AS as a powerful tool for both research and clinical applications in transfusion medicine.