De novo assembly of complete Plasmodium falciparum isolate genomes using PacBio HiFi sequencing technology

Plasmodium falciparum possesses a highly structured genome with extensive sequence diversity concentrated in Variant Surface Antigen (VSA) families. These genes-var, rif, and stevor-play key roles in immune evasion and pathogenesis and are difficult to assemble using short-read sequencing technologies. Here, we applied PacBio HiFi long-read sequencing to generate high-quality de novo genome assemblies from 43 P. falciparum parasite cultures originating from community cases in The Gambia. Parasites were culture-adapted, cloned by limiting dilution where possible, and sequenced using high molecular weight DNA extracts. Assemblies from single-genotype lineages were constructed using hifiasm, producing complete chromosomal-length scaffolds with high base accuracy without requiring short-read polishing. We recovered full repertoires of var, rif, and stevor genes and classified them into known subgroups. Together, our results demonstrate that PacBio HiFi sequencing enables accurate assembly of complex P. falciparum genomes from natural infections. This work provides a valuable genomic resource for future studies of parasite evolution, transmission dynamics, and antigenic diversity, and suggests that VSA repertoires can serve as reliable proxies of genetic relatedness across infections.