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Pooled deep sequencing of Plasmodium falciparum isolates: an efficient and scalable tool to quantify prevailing malaria drug-resistance genotypes.

Authors
  • Taylor, Steve M
  • Parobek, Christian M
  • Aragam, Nash
  • Ngasala, Billy E
  • Mårtensson, Andreas
  • Meshnick, Steven R
  • Juliano, Jonathan J
Type
Published Article
Journal
The Journal of Infectious Diseases
Publisher
Oxford University Press
Publication Date
Dec 15, 2013
Volume
208
Issue
12
Pages
1998–2006
Identifiers
DOI: 10.1093/infdis/jit392
PMID: 23908494
Source
Medline
Keywords
License
Unknown

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.

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