Development and Optimization of a Selective Whole-Genome Amplification To Study Plasmodium ovale Spp.

Abstract

Plasmodium ovale spp. has the ability to cause relapse, defined as recurring asexual parasitemia originating from liver-dormant forms. Whole-genome sequencing (WGS) data are of importance to identify putative molecular markers associated with relapse or other virulence mechanisms. Since 2010, the human-infecting malaria parasite Plasmodium ovale spp. has been divided into two genetically distinct species, P. ovale wallikeri and P. ovale curtisi . In recent years, application of whole-genome sequencing (WGS) to P. ovale spp. allowed to get a better understanding of its evolutionary history and discover some specific genetic patterns. Nevertheless, WGS data from P. ovale spp. are still scarce due to several drawbacks, including a high level of human DNA contamination in blood samples, infections with commonly low parasite density, and the lack of robust in vitro culture. Here, we developed two selective whole-genome amplification (sWGA) protocols that were tested on six P. ovale wallikeri and five P. ovale curtisi mono-infection clinical samples. Blood leukodepletion by a cellulose-based filtration was used as the gold standard for intraspecies comparative genomics with sWGA. We also demonstrated the importance of genomic DNA preincubation with the endonuclease McrBC to optimize P. ovale spp. sWGA. We obtained high-quality WGS data with more than 80% of the genome covered by ≥5 reads for each sample and identified more than 5,000 unique single-nucleotide polymorphisms (SNPs) per species. We also identified some amino acid changes in pocdhfr and powdhfr for which similar mutations in P. falciparum and P. vivax are associated with pyrimethamine or cycloguanil resistance. In conclusion, we developed two sWGA protocols for P. ovale spp. WGS that will help to design much-needed large-scale P. ovale spp. population studies. IMPORTANCE Plasmodium ovale spp. has the ability to cause relapse, defined as recurring asexual parasitemia originating from liver-dormant forms. Whole-genome sequencing (WGS) data are of importance to identify putative molecular markers associated with relapse or other virulence mechanisms. Due to low parasitemia encountered in P. ovale spp. infections and no in vitro culture available, WGS of P. ovale spp. is challenging. Blood leukodepletion by filtration has been used, but no technique exists yet to increase the quantity of parasite DNA over human DNA when starting from genomic DNA extracted from whole blood. Here, we demonstrated that selective whole-genome amplification (sWGA) is an easy-to-use protocol to obtain high-quality WGS data for both P. ovale spp. species from unprocessed blood samples. The new method will facilitate P. ovale spp. population genomic studies.