Sarah AuburnJutta MarfurtGareth MaslenSusana CampinoValentin Ruano RubioMagnus ManskeBarbara MacHunterEnny KenangalemRintis NoviyantiLeily TriantyBoni SebayangGrennady WirjanataKanlaya SriprawatDaniel AlcockBronwyn MacInnisOlivo MiottoTaane G. ClarkBruce RussellNicholas M. AnsteyFrançois NostenDominic P. KwiatkowskiRic N. PriceMenzies School of Health ResearchWellcome Trust Sanger InstituteUniversity of OxfordPapuan Health and Community Development FoundationEijkman Institute for Molecular BiologyShoklo Malaria Research UnitMahidol UniversityLondon School of Hygiene & Tropical MedicineYong Loo Lin School of MedicineRoyal Darwin HospitalNuffield Department of Clinical Medicine2018-10-192018-10-192013-01-04PLoS ONE. Vol.8, No.1 (2013)193262032-s2.0-84871885956https://repository.li.mahidol.ac.th/handle/20.500.14594/31089Whole genome sequencing (WGS) of Plasmodium vivax is problematic due to the reliance on clinical isolates which are generally low in parasitaemia and sample volume. Furthermore, clinical isolates contain a significant contaminating background of host DNA which confounds efforts to map short read sequence of the target P. vivax DNA. Here, we discuss a methodology to significantly improve the success of P. vivax WGS on natural (non-adapted) patient isolates. Using 37 patient isolates from Indonesia, Thailand, and travellers, we assessed the application of CF11-based white blood cell filtration alone and in combination with short term ex vivo schizont maturation. Although CF11 filtration reduced human DNA contamination in 8 Indonesian isolates tested, additional short-term culture increased the P. vivax DNA yield from a median of 0.15 to 6.2 ng μl-1packed red blood cells (pRBCs) (p = 0.001) and reduced the human DNA percentage from a median of 33.9% to 6.22% (p = 0.008). Furthermore, post-CF11 and culture samples from Thailand gave a median P. vivax DNA yield of 2.34 ng μl-1pRBCs, and 2.65% human DNA. In 22 P. vivax patient isolates prepared with the 2-step method, we demonstrate high depth (median 654X coverage) and breadth (≥89%) of coverage on the Illumina GAII and HiSeq platforms. In contrast to the A+T-rich P. falciparum genome, negligible bias was observed in coverage depth between coding and non-coding regions of the P. vivax genome. This uniform coverage will greatly facilitate the detection of SNPs and copy number variants across the genome, enabling unbiased exploration of the natural diversity in P. vivax populations. © 2013 Auburn et al.Mahidol UniversityAgricultural and Biological SciencesBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1371/journal.pone.0053160