Khoswe, S. | Search bioRxiv

bioRxiv2018

Resolving within-host malaria parasite diversity using single-cell sequencing

Malaria patients can carry one or more clonal lineage of the parasite, Plasmodium falciparum, but the composition of these infections cannot be directly inferred from bulk sequence data. Well-defined, complete haplotypes at single-cell resolution are ideal for describing within-host population structure and unambiguously determining parasite diversity, transmission dynamics and recent ancestry but have not been analyzed on a large scale. We generated 485 near-complete single-cell genome sequences isolated from fifteen P. falciparum patients from Chikhwawa, Malawi, an area of intense malaria transmission. Matched single-cell and bulk genomic analyses revealed patients harbored up to seventeen unique lineages. Estimation of parasite relatedness within patients suggests superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Our single-cell analysis indicates strong barriers to establishment of new infections in malaria-infected patients and allows high resolution dissection of intra-host variation in malaria parasites.

genetics↗

bioRxiv2017

High-resolution single-cell sequencing of malaria parasites

Single-cell genomics is a powerful tool for determining the genetic structure of complex communities of unicellular organisms. Patients infected with the malaria-causing parasite, Plasmodium falciparum, often carry multiple, genetically distinct parasites. Little is known about the diversity and relatedness of these lineages. We have developed an improved single-cell genomics protocol to reconstruct individual haplotypes from infections, a necessary step in uncovering parasite ecology within the host. This approach captures singly-infected red blood cells (iRBCs) by fluorescence-activated cell sorting (FACS) prior to whole genome amplification (WGA) and whole genome sequencing (WGS). Here, we demonstrate that parasites in late cell cycle stages, which contain increased DNA content, are far superior templates for generating high quality genomic data. Targeting of these cells routinely generates near-complete capture of the 23Mb P. falciparum genome (mean breadth of coverage 90.7%) at high efficiency. We used this approach to analyze the genomes of 48 individual cells from a polyclonal malaria infection sampled in Chikhwawa, Malawi. This comprehensive dataset enabled high-resolution estimation of the clonality and the relatedness of parasite haplotypes within the infection, long-standing problems in malaria biology.

genomics↗