title: Overlap Extension Barcoding for the Next Generation Sequencing and Genotyping of Plasmodium falciparum in Individual Patients in Western Kenya.
abstract: Large-scale molecular epidemiologic studies of Plasmodium falciparum parasites have provided insights into parasite biology and transmission, can identify the spread of drug resistance, and are useful in assessing vaccine targets. The polyclonal nature infections in high transmission settings is problematic for traditional genotyping approaches. Next-generation sequencing (NGS) approaches to parasite genotyping allow sensitive detection of minority variants, disaggregation of complex parasite mixtures, and scalable processing of large samples sets. Therefore, we designed, validated, and applied to field parasites an approach that leverages sequencing of individually barcoded samples in a multiplex manner. We utilize variant barcodes, invariant linker sequences and modular template-specific primers to allow for the simultaneous generation of high-dimensional sequencing data of multiple gene targets. This modularity permits a cost-effective and reproducible way to query many genes at once. In mixtures of reference parasite genomes, we quantitatively detected unique haplotypes comprising as little as 2% of a polyclonal infection. We applied this genotyping approach to field-collected parasites collected in Western Kenya in order to simultaneously obtain parasites genotypes at three unlinked loci. In summary, we present a rapid, scalable, and flexible method for genotyping individual parasites that enables molecular epidemiologic studies of parasite evolution, population structure and transmission.
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Brandt
authors.initials: B
authors.lastname: Levitt
authors.affiliation: Moi University School of Medicine, College of Health Sciences, Nandi Rd, Eldoret, Kenya.
authors.forename: Andrew
authors.initials: A
authors.lastname: Obala
authors.affiliation: Duke University DNA Analysis Facility, Department of Immunology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Langdon
authors.affiliation: Duke Center for Genomic and Computational Biology, Duke University Medical Center, Durham NC, USA.
authors.forename: David
authors.initials: D
authors.lastname: Corcoran
authors.affiliation: Division of Infectious Diseases and Duke Global Health Institute, Duke University Medical Center, Durham, NC, USA.
authors.forename: Wendy Prudhomme
authors.initials: WP
authors.lastname: O'Meara
authors.affiliation: Division of Infectious Diseases and Duke Global Health Institute, Duke University Medical Center, Durham, NC, USA.
authors.forename: Steve M
authors.initials: SM
authors.lastname: Taylor
mesh_terms: D058893:DNA Barcoding, Taxonomic; D060005:Genotyping Techniques; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016778:Malaria, Falciparum / Q000235:genetics*; D010963:Plasmodium falciparum / Q000235:genetics*; D011110:Polymorphism, Genetic