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Conventional assays often fall short, as they do not capture the wide range of allele diversity found at the <jats:italic>CYP2D6<\/jats:italic> locus, particularly in populations from malaria-endemic regions. In response, we developed a methodology integrating cost-effective DNA library preparation with nanopore sequencing, reducing reliance on extensive third-party enzymes and reagents and shortening processing times to enhance accessibility. Using well-characterized DNA standards, we evaluated various bioinformatics tools for their ability to accurately call <jats:italic>CYP2D6<\/jats:italic> star alleles. Nanopore sequencing proved particularly valuable for identifying complex structural variants, which led us to propose a novel approach to manage hybrid structural variations using long-read amplicon data. We conducted an in-depth genotypic analysis of DNA samples from 90 individuals in Thailand\u2019s Greater Mekong Subregion, achieving a high level of resolution in <jats:italic>CYP2D6<\/jats:italic> genotyping. This analysis not only identified structural variants but also a previously unidentified allele. The findings suggest potential paths forward for testing that is sensitive to local genetic variability and supports informed policy decision-making in real time, thereby potentially enhancing the effectiveness of <jats:italic>P. vivax<\/jats:italic> elimination.<\/jats:p>","DOI":"10.1038\/s41598-025-05065-2","type":"journal-article","created":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T13:35:57Z","timestamp":1759239357000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Enhancing CYP2D6 genotyping with nanopore sequencing to address allele diversity P. vivax malaria elimination"],"prefix":"10.1038","volume":"15","author":[{"given":"Thidathip","family":"Wongsurawat","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Piroon","family":"Jenjaroenpun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kanokon","family":"Suwannasin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natnicha","family":"Wankaew","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naphat","family":"Sanguansakpakdee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thananya","family":"Amornpornviwat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arjen M.","family":"Dondorp","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas P. 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MUTM2012-045-12, MU2019-025). The study received ethical approval from the review committees of the Faculty of Tropical Medicine at Mahidol University (approval no. MUTM2012-045-12, MU2019-025). This ensures that all experiments adhered to relevant guidelines and regulations, with informed consent obtained from all participants, and were conducted in accordance with the Declaration of Helsinki (98).","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"The authors declare no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"33842"}}