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Effective Preparation of Plasmodium vivax Field Isolates for High-Throughput Whole Genome Sequencing
- Source :
- PLoS ONE, PloS one, PLoS ONE, Vol 8, Iss 1, p e53160 (2013)
- Publication Year :
- 2013
- Publisher :
- Public Library of Science (PLoS), 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(-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.
- Subjects :
- 030231 tropical medicine
Plasmodium vivax
lcsh:Medicine
Genomics
Biology
Real-Time Polymerase Chain Reaction
Microbiology
Genome
DNA sequencing
03 medical and health sciences
0302 clinical medicine
parasitic diseases
Malaria, Vivax
Parasitic Diseases
Humans
Plasmodium Vivax
Genomic library
Genome Sequencing
lcsh:Science
030304 developmental biology
Whole genome sequencing
Genetics
0303 health sciences
Multidisciplinary
lcsh:R
High-Throughput Nucleotide Sequencing
Computational Biology
Plasmodium falciparum
DNA, Protozoan
biology.organism_classification
DNA extraction
Malaria
3. Good health
Plasmodium Falciparum
Infectious Diseases
Medicine
lcsh:Q
Parasitology
Genome, Protozoan
Research Article
Neglected Tropical Diseases
Subjects
Details
- ISSN :
- 19326203
- Volume :
- 8
- Database :
- OpenAIRE
- Journal :
- PLoS ONE
- Accession number :
- edsair.doi.dedup.....e21b29a4801fbfaa3a6864f5294bbf83