Single-cell genomics for dissection of complex malaria infections

Abstract

Most malaria infections contain complex mixtures of distinct parasite lineages. These multiple-genotype infections (MGIs) impact virulence evolution, drug resistance, intra-host dynamics, and recombination, but are poorly understood. To address this we have developed a single-cell genomics approach to dissect MGIs. By combining cell sorting and wholegenome amplification (WGA), we are able to generate high-quality material from parasite-infected red blood cells (RBCs) for genotyping and next-generation sequencing. We optimized our approach through analysis of >260 single-cell assays. To quantify accuracy, we decomposed mixtures of known parasite genotypes and obtained highly accurate (>99%) singlecell genotypes. We applied this validated approach directly to infections of two major malaria species, Plasmodium falciparum, for which long term culture is possible, and Plasmodium vivax, for which no long-term culture is feasible. We demonstrate that our single-cell genomics approach can be used to generate parasite genome sequences directly from patient blood in order to unravel the complexity of P. vivax and P. falciparum infections. These methods open the door for large-scale analysis of within-host variation of malaria infections, and reveal information on relatedness and drug resistance haplotypes that is inaccessible through conventional sequencing of infections.

Population, genetic, and antigenic diversity of the apicomplexanEimeria tenellaand their relevance to vaccine development

“…To shed light on the frequency of cross-fertilization events, we carried out single-sporocyst genotyping on the progeny of a series of experimental biclonal infections and identified multiple hybrid haplotypes from every one of these infections. We used the sporocyst, which contains two identical haploid genomes, as the biological unit for this work to allow FACS isolation of single-clonal units after the completion of meiosis, reducing the risk of allelic drop out (49,50).…”

Do hypnozoites cause relapse in malaria?

“…This would occasionally result in inaccurate conclusions being drawn in some epidemiological and other P. vivax studies. Whole-genome analysis and single-cell genomics should help to characterize parasites more precisely [17,80]. Thus, I consider it possible that relapse-like P. vivax malarial recurrences might have a dual origin, in other words be initiated either by hypnozoites (especially in some instances of heterologous recurrence) or nonhypnozoites that are extrahepatic but do not necessarily occur in the bloodstream [6,23,24].…”

Population genomic structure and adaptation in the zoonotic malaria parasite Plasmodium knowlesi

“…Although these data are difficult to obtain because of the generally low densities of parasites within the blood of these animals and because most macaques are infected with multiple Tables S4 and S5. species of Plasmodium (11, 32), a promising approach may be flow cytometric sorting of individual parasites for single-cell sequencing, as recently applied to human malaria parasites (33). In addition, future culture adaptation of P. knowlesi clones from the divergent genotypic clusters and the application of high-efficiency transfection technologies to study gene functions systematically could enable the investigation of phenotypic differences.…”