Open Access Highly Accessed Research article

Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

Paul Hunt12*, Axel Martinelli13, Katarzyna Modrzynska1, Sofia Borges4, Alison Creasey1, Louise Rodrigues3, Dario Beraldi105, Laurence Loewe6, Richard Fawcett1, Sujai Kumar7, Marian Thomson7, Urmi Trivedi7, Thomas D Otto8, Arnab Pain89, Mark Blaxter57 and Pedro Cravo3

Author Affiliations

1 Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

2 Centre for Immunity, Infection and Evolution, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

3 Centro de Malaria e Outras Doenças Tropicais/IHMT/UEI Biologia Molecular, Universidade Nova de Lisboa, Lisbon, Portugal

4 Centro de Malaria e Outras Doenças Tropicais/IHMT/UEI Malaria, Universidade Nova de Lisboa, Lisbon, Portugal

5 Institute for Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

6 Centre for Systems Biology at Edinburgh, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

7 The GenePool, School of Biological Sciences, University of Edinburgh, Edinburgh, UK

8 Pathogen Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, UK

9 Computational Bioscience Research Center, Chemical and Life Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia

10 Division of Genetics and Genomics, The Roslin Institute and R(D)SVS, University of Edinburgh, Roslin, UK

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BMC Genomics 2010, 11:499  doi:10.1186/1471-2164-11-499

Published: 16 September 2010

Additional files

Additional file 1:

Inventory of pyrosequencing assays. The approximate genome position (column M) of pyrosequencing assays (columns B-G) are based upon mapping to a previous assembly and annotation of AS-WTSI, using estimated lengths for telomeres, gaps between contigs etc. The locus designation pcxxpfyy-zzzz indicates that the marker maps approximately to P. chabaudi chromosome xx and homologous to a locus in P. falciparum zzzz kb along chromosome yy

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Additional file 2:

Statistics: methods, analysis and evaluation. This file describes the statistical methods developed for evaluating whether selection at given loci is significant. Two methods are described and evaluated.

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Additional file 3:

Simulations - examples. Four independent examples (#1, 9, 10, 12) of simulated genome scans of comparative index (marker (AJ, drug-sensitive parent) proportion in drug-treated population/ marker proportion in untreated population) v. genome positions of markers on 14 chromosomes (numbers at top) assuming null hypothesis of 10 resistance (AS advantage, red dots) and 10 fitness (AJ advantage, blue dots) loci are shown. The z-score (Additional File 2) of the deepest valley is calculated (bottom left). Additional File 4 plots the frequency of these z-scores after 500 such simulations. Note that, in contrast to experimental scans (Figure 3A), many simulations show multiple valleys, including minor valleys containing only neutral markers.

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Additional file 4:

Simulations - null distribution histogram. The distribution of the lowest z-scores was observed in 500 simulations of selection of the AS-30CQ × AJ cross, using experimental marker positions. The null hypothesis assumed 10 loci for artemisinin resistance and 10 minor fitness alleles. The red point indicates the experimentally observed z-score (-2.364) for chr2 under artemisinin selection (Figure 3A) corresponding to a p-value of 0.0263. See Additional File 2 for details.

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Additional file 5:

Simulations - the inferred significance of observed deepest selection valleys. These data are derived from the simulation approach. See Additional File 2 for full details. 1 z-score observed in the experiments; 2 p-value corresponding to the minimum z-score obtained experimentally; 3 simulations for ART reproduce the experiment that led to the genome-wide scan in Figure 3A.

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Additional file 6:

Mann-Whitney analysis of genome-wide LGS. Scan showing allele frequencies (proportion of AJ allele) from pyrosequencing after untreated infection (control, black triangles) and treated infection (artemisinin 100 mg/kg/day, 3 days, red diamonds) (n.b Figure 3A in main text uses Comparative Index (y-axis, defined in legend Figure 3) rather than allele proportion used here). Treatment is expected to bias AJ allele frequencies downward by removing the sensitive parent, AJ and recombinants free from resistance mutations. To correct for this and other factors (Text S1), control allele frequencies were reduced before testing potential selection valleys. Significant valleys are highlighted by blue circles (P < 0.001 in Mann-Whitney U-tests comparing a sliding window of 3-5 markers on the same chromosome (treated) with all untreated markers reduced by the percentage (R) indicated (AF-reduction); grey triangles show untreated frequencies resulting from a -25% shift). Positions of this strain's new mutations found by genome comparisons are indicated at the bottom (x = SNP; circle = indel). Chromosome numbers are given at the top. Genome position indicates physical mapping (Kbp) of 92 markers in the genome of P. chabaudi (Additional File 1). Data points are each the mean of 3 independent determinations.

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Additional file 7:

Solexa whole-genome re-sequencing - extended methods, results, analysis, evaluation and quality control. This file includes an extended description of the analysis of the Solexa Illumina data.

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Additional file 8:

Point Mutations (shared by AS-30CQ and AS-15MF wrt AS-sens). This table specifies details of point mutations predicted to be common to AS-30CQ and AS-15MF.

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Additional file 9:

Indels and CNVs (shared by AS-30CQ and AS-15MF wrt AS-sens). This table specifies details of indels and CNVs predicted to be common to AS-30CQ and AS-15MF.

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Additional file 10:

Artemisinin, pyrimethamine responses and ubp1, dhfr mutations in clones of the AS lineage of P. chabaudi. This table summarises the pyrimethamine and artemisinin responses of various clones of the lineage and their genotype, with respect to dhfr and ubp1.

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Additional file 11:

AS-15CQ genotype and the origin of alternative ubp1 V2697F mutation in AS-ATN. This file discusses a number of issues regarding AS-15CQ, its non-clonality and the origins of two mutations in ubp1.

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Additional file 12:

Validation of simulation - comparing simulation data and experimental data. This file summarises data which validates the simulation approach to statistical analysis.

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