title: Pooled sequencing and rare variant association tests for identifying the determinants of emerging drug resistance in malaria parasites.
abstract: We explored the potential of pooled sequencing to swiftly and economically identify selective sweeps due to emerging artemisinin (ART) resistance in a South-East Asian malaria parasite population. ART resistance is defined by slow parasite clearance from the blood of ART-treated patients and mutations in the kelch gene (chr. 13) have been strongly implicated to play a role. We constructed triplicate pools of 70 slow-clearing (resistant) and 70 fast-clearing (sensitive) infections collected from the Thai-Myanmar border and sequenced these to high (∼ 150-fold) read depth. Allele frequency estimates from pools showed almost perfect correlation (Lin's concordance = 0.98) with allele frequencies at 93 single nucleotide polymorphisms measured directly from individual infections, giving us confidence in the accuracy of this approach. By mapping genome-wide divergence (FST) between pools of drug-resistant and drug-sensitive parasites, we identified two large (>150 kb) regions (on chrs. 13 and 14) and 17 smaller candidate genome regions. To identify individual genes within these genome regions, we resequenced an additional 38 parasite genomes (16 slow and 22 fast-clearing) and performed rare variant association tests. These confirmed kelch as a major molecular marker for ART resistance (P = 6.03 × 10(-6)). This two-tier approach is powerful because pooled sequencing rapidly narrows down genome regions of interest, while targeted rare variant association testing within these regions can pinpoint the genetic basis of resistance. We show that our approach is robust to recurrent mutation and the generation of soft selective sweeps, which are predicted to be common in pathogen populations with large effective population sizes, and may confound more traditional gene mapping approaches.
authors.affiliation: Texas Biomedical Research Institute, San Antonio, TX ianc@txbiomedgenetics.org tanderso@txbiomedgenetics.org.
authors.forename: Ian H
authors.initials: IH
authors.lastname: Cheeseman
authors.affiliation: Texas Biomedical Research Institute, San Antonio, TX.
authors.forename: Marina
authors.initials: M
authors.lastname: McDew-White
authors.affiliation: Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand.
authors.forename: Aung Pyae
authors.initials: AP
authors.lastname: Phyo
authors.affiliation: Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand.
authors.forename: Kanlaya
authors.initials: K
authors.lastname: Sriprawat
authors.affiliation: Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
authors.forename: François
authors.initials: F
authors.lastname: Nosten
authors.affiliation: Texas Biomedical Research Institute, San Antonio, TX ianc@txbiomedgenetics.org tanderso@txbiomedgenetics.org.
authors.forename: Timothy J C
authors.initials: TJ
authors.lastname: Anderson
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D037621:Artemisinins / Q000494:pharmacology*; D001483:Base Sequence; D004351:Drug Resistance / Q000235:genetics; D056726:Genetic Association Studies; D016678:Genome; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000187:drug effects / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; C031327:artemisinin
keywords: drug resistance; malaria; pooled sequencing; rare variants