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

Abstract

Doc number: 163

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.