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Title: Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.
Authors: Auburn, Sarah
Marfurt, Jutta
Maslen, Gareth
Campino, Susana
Rubio, Valentin Ruano
Manske, Magnus
MacHunter, Barbara
Kenangalem, Enny
Noviyanti, Rintis
Trianty, Leily
Sebayang, Boni
Wirjanata, Grennady
Kanlaya Sriprawat
Alcock ,Daniel
MacInnis, Bronwyn
Miotto, Olivo
Clark, Taane G.
Russell, Bruce
Anstey, Nicholas M.
Nosten, Franc¸ois
Kwiatkowski, Dominic P.
Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Research Unit.
Mahidol University. Faculty of Tropical Medicine. Shoklo Malaria Research Unit.
Auburn, Sarah
Keywords: Genome, Protozoan;Humans;Malaria, Vivax;Open Access article
Issue Date: 2013
Citation: Auburn S, Marfurt J, Maslen G, Campino S, Ruano Rubio V, Manske M, et al. Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing. PLoS One. 2013;8(1):e53160.
Abstract: Whole 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(-1) packed 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(-1) pRBCs, 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.
ISSN: 1932-6203 (electronic)
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