Enhancing whole genome DNA amplification of Plasmodium falciparum for advanced molecular surveillance in malaria...

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. However, sequencing submicroscopic and asymptomatic cases presents challenges due to their low parasite densities and relative abundance of human DNA. Selective whole genome amplification (SWGA) is a method that employs oligonucleotide primers and phi29 DNA polymerase to specifically amplify Plasmodium DNA. SWGA has proven effective in generating increased genome coverage for various Plasmodium species. Despite its promise, current SWGA protocols have proven unsuitable in routine clinical settings. This study optimizes the SWGA protocol to improve amplification efficiency and simplify processing of clinical samples.

Thirteen P. falciparum clinical whole blood samples from returning travellers underwent SWGA with varying incubation durations of 2, 8, and 16 h. Using a newly developed SWGA protocol, three samples were additionally incubated at 3 h with EquiPhi29™ DNA Polymerase, a newer version of the phi29 enzyme. Effectiveness of the varying conditions were compared by their amplification and sequencing yield, parasite DNA recovery, genome coverage and coverage depth. After read number normalization through random selection with rasusa, pairwise SNP comparison was also performed to ensure variant calling by freebayes was unaffected by the changes in condition. Pairwise agreement was tested by Cohen's Kappa. Drug resistance profiles for each sample were generated with Malaria-Profiler to further verify these findings.

The results demonstrate that there were comparable amplification and sequencing yields between the 8 and 16 h SWGA durations. These durations also showed significant results similarities in variant calling with up to > 90% SNP concordance. Moreover, using the newer developed SWGA protocol, sequencing yields and genome coverage were significantly enhanced, achieving over 63% P. falciparum genome coverage in just three hours processing prior to sequencing. While reducing reagent costs by almost 60%. Overall, drug resistance profiles remained consistent across all tested conditions.

This advance holds promise for faster, more cost-effective malaria diagnostics and genomic surveillance, improving clinical decision-making and supporting malaria elimination efforts.