Resolving haplotypes of the glucose-6-phosphate dehydrogenase gene using long-range polymerase chain reaction and Oxford Nanopore sequencing

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy and poses a major concern on safe administration of oxidative drugs, including antimalarials such as primaquine and tafenoquine. Common diagnostic approaches, such as enzyme assays, polymerase chain reaction (PCR)-based methods, and short-read genotyping, often fail to identify heterozygous female carriers and are unable to determine the phase of compound heterozygous mutations. To address these limitations, a workflow combining long-range PCR with Oxford Nanopore Technologies (ONT) sequencing was developed, enabling comprehensive analysis of the entire G6PD locus with direct haplotype resolution. The accuracy of the developed method was independently validated by Sanger sequencing for exonic variant detection and by adaptive sampling-based ONT sequencing for phasing accuracy. A total of 24 samples (20 females, 4 males) were analyzed using two long-range amplicons (∼12 and ∼14 kb) with an 8.2 kb overlap spanning both variant-rich and variant-sparse regions. ONT sequencing revealed 36 distinct variants across exonic, intronic, and regulatory regions. The design consistently captured multiple informative heterozygous sites, markedly improving haplotype reconstruction in females. Nanopore sequencing generated long reads (N50 ∼11 kb) with deep coverage (>700-fold), supporting accurate variant detection and phasing. These findings demonstrate the feasibility and robustness of a nanopore-based long-read approach for comprehensive G6PD haplotyping, integrating variant detection and phasing within a single analytical workflow, and providing a foundation for future studies on carrier detection and other X-linked genes.