High quality chromosomal genome assemblies of three human Plasmodium species directly from natural infections

Malaria, caused by mosquito-transmitted Plasmodium parasites, remains a major global health challenge. While P. falciparum accounts for most malaria deaths, the lesser-studied P. ovale spp. and P. malariae also contribute to disease burden across many endemic regions, typically causing milder but chronic infections. Here, we generated high quality, chromosome-level reference genomes for P. ovale wallikeri, P. malariae, and P. falciparum directly from blood samples taken from natural infections. Using PacBio Ultra-Low-Input HiFi long-read sequencing and Hi-C chromatin conformation capture, we overcome limitations posed by low parasite biomass and the absence of in vitro culture for P. ovale wallikeri and P. malariae. These high quality reference genomes resolve previously inaccessible subtelomeric regions that include expanded gene families likely involved in host-parasite interactions, and they enhance our understanding of parasite persistence, transmission, immune evasion and comparative evolutionary studies. This work provides a critical foundation for including these neglected species in global malaria elimination efforts.