title: A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.
abstract: A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy.
authors.affiliation: Center for Genomics and Systems Biology, Department of Biology, New York University, New York, New York, USA.
authors.forename: Pavitra N
authors.initials: PN
authors.lastname: Rao
authors.affiliation: Center for Genomics and Systems Biology, Department of Biology, New York University, New York, New York, USA.
authors.forename: Swapna
authors.initials: S
authors.lastname: Uplekar
authors.affiliation: National Institute of Technology, Raurkela, Odisha, India.
authors.forename: Sriti
authors.initials: S
authors.lastname: Kayal
authors.affiliation: National Institute of Malaria Research, Indian Council of Medical Research, Dwarka, New Delhi, India.
authors.forename: Prashant K
authors.initials: PK
authors.lastname: Mallick
authors.affiliation: National Institute of Malaria Research, Indian Council of Medical Research, Dwarka, New Delhi, India.
authors.forename: Nabamita
authors.initials: N
authors.lastname: Bandyopadhyay
authors.affiliation: National Institute of Malaria Research, Indian Council of Medical Research, Dwarka, New Delhi, India.
authors.forename: Sonal
authors.initials: S
authors.lastname: Kale
authors.affiliation: National Institute of Malaria Research, Indian Council of Medical Research, Dwarka, New Delhi, India.
authors.forename: Om P
authors.initials: OP
authors.lastname: Singh
authors.affiliation: Institute of Life Sciences, Bhubaneswar, Odisha, India.
authors.forename: Akshaya
authors.initials: A
authors.lastname: Mohanty
authors.affiliation: Ispat General Hospital, Raurkela, Odisha, India.
authors.forename: Sanjib
authors.initials: S
authors.lastname: Mohanty
authors.affiliation: Department of Microbiology, Division of Parasitology, New York University School of Medicine, New York, New York, USA.
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Wassmer
authors.affiliation: Center for Genomics and Systems Biology, Department of Biology, New York University, New York, New York, USA jane.carlton@nyu.edu.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Carlton
mesh_terms: D000483:Alleles; D000962:Antimalarials / Q000494:pharmacology*; D019295:Computational Biology / Q000379:methods; D004351:Drug Resistance; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007194:India; D016778:Malaria, Falciparum / Q000469:parasitology*; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D000962:Antimalarials