Publication:
Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing

Issued Date

2013-01-04

Resource Type

Article

ISSN

19326203

DOI

10.1371/journal.pone.0053160

Other identifier(s)

2-s2.0-84871885956

Rights

Mahidol University

Rights Holder(s)

SCOPUS

Bibliographic Citation

PLoS ONE. Vol.8, No.1 (2013)

Suggested Citation

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 Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing. PLoS ONE. Vol.8, No.1 (2013). doi:10.1371/journal.pone.0053160 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/31089

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Title

Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing

Author(s)

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

Other Contributor(s)

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

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.

Keyword(s)

Agricultural and Biological Sciences
 Biochemistry, Genetics and Molecular Biology

Availability

URI

https://repository.li.mahidol.ac.th/handle/20.500.14594/31089

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