Whole Genome Sequencing of Plasmodium vivax from the China-Myanmar Border Using Whole Genome Sequence Capture

Background: Efforts to control Plasmodium vivax malaria are hindered by the lack of a vaccine, drug resistance and the challenge of preventing relapse and reinfections. Whole genome sequencing of clinical isolates help to overcome some of these challenges, providing insight into drug and vaccine development. However, low parasite densities in P. vivax infections along with host DNA contamination have made it difficult to achieve uniform parasite genome coverage and data quality in the past. Materials and Methods: We applied the Roche-NimbleGen SeqCap EZ whole genome sequence capture method to leukocyte-depleted clinical samples from a patient with symptomatic P. vivax malaria on the China-Myanmar border. Capture probes were designed against the entire genome assembly of P. vivax Sal I reference isolate. The gDNA library was sequenced with and without capture, and using Illumina HiSeq and MiSeq platforms, to compare the quality, breadth and depth of genome coverage of the sequence, and the suitability of the data for genome assembly. Results: Whole genome capture method was highly effective in capturing extremely low quantities of parasite DNA from a very small amount of total DNA (1.41g). Even though the depletion of leukocytes was very effective in eliminating most human DNA (90.66% of the pre-capture reads were of parasite origin), the capture method resulted in a nearly complete enrichment of parasite DNA, with >98.5% of the reads post-capture mapping to P. vivax reference genome. At 95%, the breadth of genome coverage was very high and similar pre- and post-capture, demonstrating that the probe set enables the comprehensive capture of the parasite genome. Conclusions: With close to 100% enrichment of parasite DNA, and nearly complete coverage of P. vivaxreference genome, this novel whole genome sequence capture application dramatically improves our ability to recover P. vivax DNA from clinical samples within areas having limited capacity for sample processing and cold transport, such as the China-Myanmar border region. This enhanced capability to generate whole genome sequence will improve our understanding of P. vivax genetic diversity, population structure and parasite migration patterns, potentially leading to new tools and approaches for elimination in low transmission and pre-elimination settings.