Optimisation of Plasmodium DNA enrichment to provide better sequencing capabilities.

Genomics is critical for malaria control and elimination by providing population-level monitoring of malarial gene flow. Whole genome sequencing is valuable for identifying such genomic changes and antimalarial drug resistance, which is vital as resistance compromises the effectiveness of existing therapies. However, sequencing submicroscopic and asymptomatic cases, comprising the majority of malaria-exposed populations, present challenges due to their low parasite densities and relative abundance of human DNA. Additionally, excluding these cases risks biasing genomic datasets, making them only representative of febrile disease. Selective whole genome amplification (SWGA) is a method that employs oligonucleotide primers and high-fidelity DNA polymerases to specifically amplify Plasmodium DNA and thus provide a more advantageous relative abundance of parasite:human DNA. SWGA has proven effective in generating increased genome coverage for various Plasmodium species, thus enhancing sequencing efficiency and improving the detection of genetic variants. Despite its promise, current SWGA protocols have proven unsuitable for WGS from small-volume clinical samples. This study optimises the SWGA protocol to improve amplification efficiency and simplify processing of precious clinical material. Our results demonstrate that by reducing incubation times and using a newer version of phi29 DNA polymerase we significantly enhance sequencing yields and genome coverage, with the new protocol achieving over 63% P. falciparum genome coverage in just three hours processing prior to loading onto the sequencing flowcell, while also reducing reagent costs by almost 60%. This advancement holds promise for faster, more cost-effective malaria diagnostics and genomic surveillance, improving clinical decision-making and supporting malaria elimination efforts.