Single-cell genomics for dissection of complex malaria infections
- Shalini Nair1,
- Standwell C. Nkhoma2,
- David Serre3,
- Peter A. Zimmerman4,
- Karla Gorena5,
- Benjamin J. Daniel5,
- François Nosten6,7,
- Timothy J.C. Anderson1 and
- Ian H. Cheeseman1,8
- 1Texas Biomedical Research Institute, San Antonio, Texas 78227-5301, USA;
- 2Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Chichiri, Blantyre 3, Malawi;
- 3Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio 44195, USA;
- 4Center for Global Health and Diseases, Case Western Reserve University, Cleveland, Ohio 44106, USA;
- 5University of Texas Health Science Center San Antonio, San Antonio, Texas 78229, USA;
- 6Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Tak 63110, Thailand;
- 7Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford 0X3 7LJ, United Kingdom
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 whole-genome 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%) single-cell 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.
Footnotes
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↵8 Corresponding author
E-mail ianc{at}txbiomedgenetics.org
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.168286.113.
- Received October 11, 2013.
- Accepted March 26, 2014.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
