Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing.
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 se...
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th-mahidol.6672023-03-30T10:34:24Z Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing. 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. Auburn, Sarah Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Research Unit. Mahidol University. Faculty of Tropical Medicine. Shoklo Malaria Research Unit. Genome, Protozoan Humans Malaria, Vivax Open Access article 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. 2014-06-30T07:02:26Z 2016-09-21T03:24:49Z 2014-06-30T07:02:26Z 2016-09-21T03:24:49Z 2014-06-25 2013 Article 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. 10.1371/journal.pone.0053160. 1932-6203 (electronic) https://repository.li.mahidol.ac.th/handle/123456789/667 eng Mahidol University PLOS one application/pdf |
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Genome, Protozoan Humans Malaria, Vivax Open Access article |
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Genome, Protozoan Humans Malaria, Vivax Open Access article 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. |
description |
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. |
author2 |
Auburn, Sarah |
author_facet |
Auburn, Sarah 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. |
format |
Article |
author |
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. |
author_sort |
Auburn, Sarah |
title |
Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing. |
title_short |
Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing. |
title_full |
Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing. |
title_fullStr |
Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing. |
title_full_unstemmed |
Effective preparation of Plasmodium vivax field isolates for high-throughput whole genome sequencing. |
title_sort |
effective preparation of plasmodium vivax field isolates for high-throughput whole genome sequencing. |
publishDate |
2014 |
url |
https://repository.li.mahidol.ac.th/handle/123456789/667 |
_version_ |
1763494620464939008 |