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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

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

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Anne Boissière, Céline Arnathau, Christophe Duperray, Laurence Berry, Laurence Lachaud, François Renaud, Patrick Durand, Franck Prugnolle
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-163) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AB, CA, PD, FR, FP designed the study. AB, CA, CD, PD, LL, LB performed the experiments. AB, CA, PD, CD, FR, FP analysed the data. All authors put forward different ideas and contributed to the early draft. AB, CA, FP wrote the paper. All authors agreed the final draft. *AB and CA contributed equally to this work. All authors read and approved the final manuscript.

Abstract

Background

Flow 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.

Methods

The 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 conclusion

The 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.

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Zusatzmaterial
Authors’ original file for figure 1
12936_2011_2148_MOESM1_ESM.pdf
Authors’ original file for figure 2
12936_2011_2148_MOESM2_ESM.pdf
Literatur
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