Publication: Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.
Accepted Date
2012-11-26
Issued Date
2013
Resource Type
Language
eng
ISSN
1932-6203 (electronic)
Rights
Mahidol University
Rights Holder(s)
PLOS one
Bibliographic 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.
Suggested Citation
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. Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.. 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.. doi:10.1371/journal.pone.0053160. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/667
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Title
Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.
Author(s)
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.
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.
Corresponding Author(s)
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.