Open Access Research article

Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria

Katarzyna Kinga Modrzynska18, Alison Creasey1, Laurence Loewe2, Timothee Cezard3, Sofia Trindade Borges49, Axel Martinelli5, Louise Rodrigues105, Pedro Cravo115, Mark Blaxter36, Richard Carter1 and Paul Hunt17*

Author Affiliations

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

2 Laboratory of Genetics and Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, USA

3 The GenePool, University of Edinburgh, Edinburgh, UK

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

5 Centro de Malaria e Outras Doenças Tropicais/IHMT/UEI Biologia Molecular, Lisbon, Portugal

6 Institute for Evolutionary Biology, University of Edinburgh, Edinburgh, UK

7 Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK

8 Current address: The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK

9 Current address: Research Unit and Cardiology department, Funchal Hospital Center, Funchal. Madeira, Portugal

10 Current address: Microbiology, Molecular Genetics and Immunology, Kansas University Medical Center, Kansas City, USA

11 Current address: IPTSP, Universidade Federal de Goiás, Goiânia, Brasil

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BMC Genomics 2012, 13:106  doi:10.1186/1471-2164-13-106

Published: 21 March 2012

Additional files

Additional file 1:

Additional Text. Section 1, Solexa genome re-sequencing; Section 2, Other mutations in AS-30CQ; Section 3, AS-15CQ and the origins of different haplotypes in subsequent clones; Section 4, Discontinuities in AJ allele frequency.

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Additional file 2:

(Table) Solexa whole-genome re-sequencing metrics.

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Additional file 3:

(Figure) LGS-pyro v LGS-Illumina. Comparison of genome scans (LGS-pyro (top), LGS-Illumina (bottom)) show near perfect correspondence between the two methodologies. Vertical axis (linear) indicates proportion of AJ alleles in parasites surviving 3 mg CQ kg-1 day-1. Horizontal axis indicates chromosome number, top and bottom or genome co-ordinate (Kbase), top only. Position of mutations (AS-30CQ relative to AS-sens) are indicated at bottom of bottom panel (7 SNPs x, 2 deletions •).

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Additional file 4:

(Table) AS-30CQ Genome re-sequencing. Summary of all the mutations proposed in clone AS-30CQ (Additional File 1). Highlighted are mutations confirmed by di-deoxy sequencing (green), rejected mutations (red), a high confidence deletion (yellow) and low confidence mutations (orange). Read depth according to SSAHA2 is provided for SNPs. All quality scores for SNPs were according to SSAHA2. Small indel quality scores indicate the number of reads calling an indel divided by the total number of reads covering the indel. For large indels and CNVs, a comparative coverage was calculated as described (Methods section and Additional File 1).

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Additional file 5:

(Table) Genome-wide analysis of % of bases with read-coverage ≥ 10.

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Additional file 6:

(Figure) The appearance of mutations in the AS lineage. Mutations are described by chromosomal location, gene ID, specific amino acid change etc. Some were previously described [29,30] (blue). Novel mutations are identified here (red). For both PCHAS_030137 and ubp1, alternative mutations arising between AS-3CQ and AS-15CQ (and individually selected in AS-15MF [31,35] and AS-ATN [55] during mefloquine and artesunate selection, respectively) are defined (a-d). Refer to Additional File 1(section 3) for further details.

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Additional file 7:

(Figure) Chromosome 7 - 34 bp deletion. Close to the 3' end of gene PCHAS_072420, an alignment of nucleotide sequences for reference genome sequence (AS-WTSI), the sensitive AS lineage progenitor (AS-sens) and the drug resistant mutants AS-PYR, AS-3CQ, AS-30CQ and AS-ART is shown. Symbols: -,34 bp deletion in AS-PYR and subsequent clones (wrt nt 197 - 230 (AS-WTSI arbitrary numbering) inclusive), 15 bp deletion in AS strains (wrt nt 306 - 320 (AJ arbitrary numbering) inclusive); *, nucleotides identical in all clones and strains investigated here (note high frequency of AS/AJ SNPs). 3' end of coding sequence of gene PCHAS_072420 is indicated (upper case, green highlighting); intron (lower case, grey highlighting), 3'-UT and intergenic region indicated (lower case); termination codon (red). Repetitive sequences which may mediate the 34 bp and 15 bp deletions are indicated (yellow) in individual representative clones.

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Additional file 8:

(Table) Discontinuity co-ordinates. Co-ordinates represent nucleotide position on chromosome relative to the AS-WTXI sequence assembly (Sanger Sept 2009); nd, cannot be determined.

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Additional file 9:

(Table) Primers used. These oligonucleotide primers were used to confirm the predicted mutations in AS lineage. Pairs of primers marked with * were also used for proportional sequencing.

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