Isolation of Plasmodium falciparum by flow-cytometry: implications for single-trophozoite genotyping and parasite DNA purification for whole-genome high-throughput sequencing of archival ... View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2012-05-14

AUTHORS

Anne Boissière, Céline Arnathau, Christophe Duperray, Laurence Berry, Laurence Lachaud, François Renaud, Patrick Durand, Franck Prugnolle

ABSTRACT

BackgroundFlow cytometry and cell sorting are powerful tools enabling the selection of particular cell types within heterogeneous cell mixtures. These techniques, combined with whole genome amplification that non-specifically amplify small amounts of starting DNA, offer exciting new opportunities for the study of malaria genetics. Among them, two are tested in this paper: (1) single cell genotyping and (2) parasite DNA purification for subsequent whole genome sequencing using shotgun technologies.MethodsThe method described allows isolation of Plasmodium falciparum trophozoites, genotyping and whole genome sequencing from the blood of infected patients. For trophozoite isolation, parasite and host nuclei are stained using propidium iodide (PI) followed by flow cytometry and cell sorting to separate trophozoites from host cells. Before genotyping or sequencing, whole genome amplification is used to increase the amount of DNA within sorted samples. The method has been specifically designed to deal with frozen blood samples.Results and conclusionThe results demonstrate that single trophozoite genotyping is possible and that cell sorting can be successfully applied to reduce the contaminating host DNA for subsequent whole genome sequencing of parasites extracted from infected blood samples. More... »

PAGES

163

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1475-2875-11-163

DOI

http://dx.doi.org/10.1186/1475-2875-11-163

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1041241671

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/22583664


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