Enhancing CYP2D6 genotyping with nanopore sequencing to address allele diversity P. vivax malaria elimination

Abstract

The precise profiling of CYP2D6 alleles is critical for identifying patients who are likely to benefit from primaquine therapy, a standard treatment for preventing P. vivax relapse and, as a result, obstacle for malaria elimination. Conventional assays often fall short, as they do not capture the wide range of allele diversity found at the CYP2D6 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 CYP2D6 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's Greater Mekong Subregion, achieving a high level of resolution in CYP2D6 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 P. vivax elimination.