Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

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Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance1,2. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P. falciparum genome.

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Figure 1: Allele frequency spectrum of SNPs genotyped in this study.
Figure 2: Representations of a pairwise distance matrix between the 227 samples analysed.
Figure 3: Quantification of within-host diversity.

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Data deposits

All sequence data are available online at the European Nucleotide Archive (ENA); accession numbers are listed in Supplementary Table 12. An online catalogue of SNPs and allele frequencies is available at


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We thank G. Dougan and N. Day for support, and T. Anderson and M. Mackinnon for comments. The sequencing and analysis components of this study were supported by the Wellcome Trust through Sanger Institute core funding (077012/Z/05/Z; 098051) and a Strategic Award (090770/Z/09/Z); the Medical Research Council (MRC) through the MRC Centre for Genomics and Global Health (G0600718) and an MRC Professorship to D.P.K. (G19/9). Other parts of this study were partly supported by the Wellcome Trust including core support to the Wellcome Trust Centre for Human Genetics (075491/Z/04; 090532/Z/09/Z); the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health; and a Howard Hughes Medical Institute International Scholarship (55005502) to A.D.

Author information

S.A., S.C., A.D., O.D., I.Z., J.-B.O., P.M., I.M., P.S., A.N., S.B., S.M.K., K.M., H.J., X.-Z.S., C.A., R.F., D.S., F.N., M.I., N.J.W., L.A.-E., C.S., V.M., D.M., A.A.-N. and D.J.C. performed field and laboratory studies to obtain P. falciparum samples for sequencing. S.A., S.C., M.S., E.A., D.A., E.D., S.O., M.A.Q., D.J.T., B.M., C.I.N. and M.B. developed and implemented methods for sample processing and sequencing library preparation. J.A.-G., M.M., O.M., G.M., V.R.R. and D.J. developed software for data management and visualization. K.A.R., C.H., A.J., K.R., J.C.T., M.T.F., S.C., S.A., D.A., C.I.N. and M.B. performed validation experiments. C.V.P., S.T.-H. and C.R. contributed to development of the project. B.M., M.B., C.I.N. and J.C.R. provided project management and oversight. O.M., M.M., D.P.K., J.O.’B. and T.G.C. conducted data analyses. D.P.K. and O.M. developed the Fws metric. D.P.K., O.M. and M.M. wrote the manuscript and collated comments from all authors. S.A. and S.C. made equal contributions.

Correspondence to Dominic P. Kwiatkowski.

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The authors declare no competing financial interests.

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Supplementary Information

This file contains Supplementary Methods, which include references, tables 1-5 and figures 1-9, Supplementary Tables 1-12 and Supplementary Figures 1-18 (see page 1 for details). (PDF 4937 kb)

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Manske, M., Miotto, O., Campino, S. et al. Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature 487, 375–379 (2012) doi:10.1038/nature11174

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