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. Malaria kills over 400,000 people annually throughout the tropics 1 , and new tools are continually needed to track and type parasites to improve disease control. Plasmodium falciparum, the deadliest and most virulent malaria parasite species, has a large and highly diverse genome 2 . Population genetics offers a research toolset to elucidate the parasite’s transmission patterns and define and track medically-important genotypes such as those respon- sible for more severe disease or conferring drug- or vaccine-resistance 3–5 . Recent advances in next generation sequencing (NGS) technology have broadened the utility of these approaches 6 , allowing more comprehensive detection of parasite genotypes present in complex parasitemias 7 . In doing so, these approaches can enable better understanding of minority variants including their transmission 8 , contribution to parasite population structure, and involvement in drug and vaccine candidate resistance 9 .