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Title: Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing
Authors: Sarah Auburn
Jutta Marfurt
Gareth Maslen
Susana Campino
Valentin Ruano Rubio
Magnus Manske
Barbara MacHunter
Enny Kenangalem
Rintis Noviyanti
Leily Trianty
Boni Sebayang
Grennady Wirjanata
Kanlaya Sriprawat
Daniel Alcock
Bronwyn MacInnis
Olivo Miotto
Taane G. Clark
Bruce Russell
Nicholas M. Anstey
François Nosten
Dominic P. Kwiatkowski
Ric N. Price
Menzies School of Health Research
Wellcome Trust Sanger Institute
University of Oxford
Papuan Health and Community Development Foundation
Eijkman Institute for Molecular Biology
Shoklo Malaria Research Unit
Mahidol University
London School of Hygiene & Tropical Medicine
Yong Loo Lin School of Medicine
Royal Darwin Hospital
Nuffield Department of Clinical Medicine
Keywords: Agricultural and Biological Sciences;Biochemistry, Genetics and Molecular Biology
Issue Date: 4-Jan-2013
Citation: PLoS ONE. Vol.8, No.1 (2013)
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-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.
ISSN: 19326203
Appears in Collections:Scopus 2011-2015

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