title: Pooled deep sequencing of Plasmodium falciparum isolates: an efficient and scalable tool to quantify prevailing malaria drug-resistance genotypes.
abstract: Molecular surveillance for drug-resistant malaria parasites requires reliable, timely, and scalable methods. These data may be efficiently produced by genotyping parasite populations using second-generation sequencing (SGS). We designed and validated a SGS protocol to quantify mutant allele frequencies in the Plasmodium falciparum genes dhfr and dhps in mixed isolates. We applied this new protocol to field isolates from children and compared it to standard genotyping using Sanger sequencing. The SGS protocol accurately quantified dhfr and dhps allele frequencies in a mixture of parasite strains. Using SGS of DNA that was extracted and then pooled from individual isolates, we estimated mutant allele frequencies that were closely correlated to those estimated by Sanger sequencing (correlations, >0.98). The SGS protocol obviated most molecular steps in conventional methods and is cost saving for parasite populations >50. This SGS genotyping method efficiently and reproducibly estimates parasite allele frequencies within populations of P. falciparum for molecular epidemiologic studies.
authors.affiliation: Department of Epidemiology, Gillings School of Global Public Health.
authors.forename: Steve M
authors.initials: SM
authors.lastname: Taylor
authors.forename: Christian M
authors.initials: CM
authors.lastname: Parobek
authors.forename: Nash
authors.initials: N
authors.lastname: Aragam
authors.forename: Billy E
authors.initials: BE
authors.lastname: Ngasala
authors.forename: Andreas
authors.initials: A
authors.lastname: Mårtensson
authors.forename: Steven R
authors.initials: SR
authors.lastname: Meshnick
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Juliano
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D001483:Base Sequence; D002648:Child; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics*; D005787:Gene Frequency; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016778:Malaria, Falciparum / Q000097:blood / Q000469:parasitology*; D017720:Molecular Epidemiology; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000145:classification / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods; D013636:Tanzania
chemical_list: D000962:Antimalarials; D016054:DNA, Protozoan
keywords: Plasmodium falciparum; drug resistance; molecular surveillance; tropical diseases
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