Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing

Article metrics

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Accession codes

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 http://www.malariagen.net/resource/10.

References

  1. 1

    Wootton, J. C. et al. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum. Nature 418, 320–323 (2002)

  2. 2

    Dondorp, A. M. et al. Artemisinin resistance in Plasmodium falciparum malaria. N. Engl. J. Med. 361, 455–467 (2009)

  3. 3

    Gardner, M. J. et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511 (2002)

  4. 4

    Mu, J. et al. Genome-wide variation and identification of vaccine targets in the Plasmodium falciparum genome. Nature Genet. 39, 126–130 (2007)

  5. 5

    Volkman, S. K. et al. A genome-wide map of diversity in Plasmodium falciparum. Nature Genet. 39, 113–119 (2007)

  6. 6

    Jeffares, D. C. et al. Genome variation and evolution of the malaria parasite Plasmodium falciparum. Nature Genet. 39, 120–125 (2007)

  7. 7

    Neafsey, D. E. et al. Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence. Genome Biol. 9, R171 (2008)

  8. 8

    Mu, J. et al. Plasmodium falciparum genome-wide scans for positive selection, recombination hot spots and resistance to antimalarial drugs. Nature Genet. 42, 268–271 (2010)

  9. 9

    Auburn, S. et al. An effective method to purify Plasmodium falciparum DNA directly from clinical blood samples for whole genome high-throughput sequencing. PLoS ONE 6, e22213 (2011)

  10. 10

    Joy, D. A. et al. Early origin and recent expansion of Plasmodium falciparum. Science 300, 318–321 (2003)

  11. 11

    Li, J. Z. et al. Worldwide human relationships inferred from genome-wide patterns of variation. Science 319, 1100–1104 (2008)

  12. 12

    Prugnolle, F. et al. African great apes are natural hosts of multiple related malaria species, including Plasmodium falciparum. Proc. Natl Acad. Sci. USA 107, 1458–1463 (2010)

  13. 13

    Anderson, T. J. et al. Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol. Biol. Evol. 17, 1467–1482 (2000)

  14. 14

    Hartl, D. & Clark, A. G. Principles of population genetics 4th edn (Sinauer, 2007)

  15. 15

    Paul, R. E. et al. Mating patterns in malaria parasite populations of Papua New Guinea. Science 269, 1709–1711 (1995)

  16. 16

    Dye, C. & Williams, B. G. Multigenic drug resistance among inbred malaria parasites. Proc. R. Soc. Lond. B 264, 61–67 (1997)

  17. 17

    Hill, W. G., Babiker, H. A., Ranford-Cartwright, L. C. & Walliker, D. Estimation of inbreeding coefficients from genotypic data on multiple alleles, and application to estimation of clonality in malaria parasites. Genet. Res. 65, 53–61 (1995)

  18. 18

    Auburn, S. et al. Characterization of within-host Plasmodium falciparum diversity using next-generation sequence data. PLoS ONE 7, e32891 (2012)

  19. 19

    Smith, D. L., Drakeley, C. J., Chiyaka, C. & Hay, S. I. A quantitative analysis of transmission efficiency versus intensity for malaria. Nature Commun. 1, 108 (2010)

  20. 20

    Trung, H. D. et al. Malaria transmission and major malaria vectors in different geographical areas of Southeast Asia. Trop. Med. Int. Health 9, 230–237 (2004)

  21. 21

    Paul, R. E. et al. Genetic analysis of Plasmodium falciparum infections on the north-western border of Thailand. Trans. R. Soc. Trop. Med. Hyg. 93, 587–593 (1999)

  22. 22

    Schultz, L. et al. Multilocus haplotypes reveal variable levels of diversity and population structure of Plasmodium falciparum in Papua New Guinea, a region of intense perennial transmission. Malar. J. 9, 336 (2010)

  23. 23

    Su, X. et al. A genetic map and recombination parameters of the human malaria parasite Plasmodium falciparum. Science 286, 1351–1353 (1999)

  24. 24

    Mehlotra, R. K. et al. Evolution of a unique Plasmodium falciparum chloroquine-resistance phenotype in association with pfcrt polymorphism in Papua New Guinea and South America. Proc. Natl Acad. Sci. USA 98, 12689–12694 (2001)

  25. 25

    van Dijk, M. R. et al. Three members of the 6-cys protein family of Plasmodium play a role in gamete fertility. PLoS Pathog. 6, e1000853 (2010)

  26. 26

    Anthony, T. G., Polley, S. D., Vogler, A. P. & Conway, D. J. Evidence of non-neutral polymorphism in Plasmodium falciparum gamete surface protein genes Pfs47 and Pfs48/45. Mol. Biochem. Parasitol. 156, 117–123 (2007)

  27. 27

    Martin, R. E., Henry, R. I., Abbey, J. L., Clements, J. D. & Kirk, K. The ‘permeome’ of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum. Genome Biol. 6, R26 (2005)

  28. 28

    Boisson, B. et al. The novel putative transporter NPT1 plays a critical role in early stages of Plasmodium berghei sexual development. Mol. Microbiol. 81, 1343–1357 (2011)

  29. 29

    Kozarewa, I. et al. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes. Nature Methods 6, 291–295 (2009)

  30. 30

    Oyola, S. O. et al. Optimizing Illumina Next-Generation Sequencing library preparation for extremely AT-biased genomes. BMC Genomics 13, 1 (2012)

Download references

Acknowledgements

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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

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)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.