Selective Whole Genome Amplification of Plasmodium malariae DNA from clinical samples reveals high genetic diversity and population structure of this neglected malaria parasite. SWGA of Plasmodium malariae

The genomic diversity of Plasmodium malariae malaria parasites is understudied, partly because infected individuals tend to present with low parasite densities, leading to difficulties in obtaining sufficient parasite DNA for genome analysis. Selective whole genome amplification (SWGA) increases the relative levels of pathogen DNA in a clinical sample, but has not been adapted for P. malariae parasites. Here we design customized SWGA primers which successfully amplify P. malariae DNA extracted directly from unprocessed clinical blood samples obtained from 9 countries. SWGA enables the successful WGS of samples with low parasite density (>0.005%), leading to an average of 4.6-fold enrichment of P. malariae DNA, and an average 14.5-fold increase in genome coverage when compared to unamplified samples. We identify 781,289 high quality genome-wide single nucleotide polymorphisms (SNPs) across 16 isolates. Population genetic analysis suggested that P. malariae parasites display geographical separation by continent. Further, SWGA successfully amplified and allowed SNP discovery in genetic regions of interest such as orthologs of P. falciparum drug resistance-associated loci (Pfcrt, Pfdhfr, Pfdhps, Pfk13 and Pfmdr1), and a high degree of polymorphism was found within subtelomeric regions, where the multivariable fam genes are located. In conclusion, we have established a robust SWGA approach that can assist the whole genome sequencing of P. malariae, and thereby facilitate the implementation of much-needed large-scale multi-population genomic studies of this neglected malaria parasite. As demonstrated in other Plasmodia, such genetic diversity studies can provide insights into the biology underlying the disease and inform malaria surveillance and control measures.