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.
Highlights
Flow cytometry and cell sorting are powerful tools enabling the selection of particular cell types within heterogeneous cell mixtures
Flow cytometry and cell sorting are powerful technologies that allow the physical separation of a cell, or a particle of interest, from a heterogeneous cell mixture
General method of isolation and applications The detailed protocol outlines trophozoite cell preparation from archived frozen samples, characterization and cell isolation. It is followed by whole-genome amplification that provides a template for single-cell microsatellite genotyping and multiple-cell whole-genome sequencing
Summary
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 nonspecifically amplify small amounts of starting DNA, offer exciting new opportunities for the study of malaria genetics. Two are tested in this paper: (1) single cell genotyping and (2) parasite DNA purification for subsequent whole genome sequencing using shotgun technologies. Flow cytometry and cell sorting are powerful technologies that allow the physical separation of a cell, or a particle of interest, from a heterogeneous cell mixture These techniques, combined with specific technologies in genetics such as whole genome amplification, provide new opportunities for malaria genetic studies. Ecological interactions between these lineages within hosts can influence the parasite’s life history traits (virulence, transmission) and can have important consequences for their epidemiology and evolution [2,3]
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