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.

High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology

“…At 10 USD per sample, the current study is approximately 7x cheaper than an alternative protocol published for Ion Torrent 41 . We have created 50 unique indices (Table S2), which make it possible to create 2450 unique dual index combinations (50 × 50–50) and hence run as many samples on the Miseq simultaneously.…”

Molecular Epidemiology of Plasmodium falciparum kelch13 Mutations in Senegal Determined by Using Targeted Amplicon Deep Sequencing

“…NGS is becoming more affordable; the application of this technology for monitoring the emergence of drug resistance (15,20) is already proving to be a useful diagnostic tool for the surveillance and early detection of changes in allele frequency in drug resistance loci within individual samples. This approach increases our understanding of the evolutionary consequences of malaria control programs (i.e., fitness cost and selection of resistance alleles under different scenarios across geographic regions).…”

“…In addition, as new gene targets are identified, they can be easily included in targeted deep amplicon sequencing approaches (20). While NGS is already used in clinical laboratory settings in the United States, such as the MiSeqDx system (30), it can also be readily be adopted as a robust and rapid drug resistance surveillance tool in sentinel sites in Africa (7).…”

“…While pooled deep sequencing is substantially less costly, it cannot provide results on individual patient levels, which are critical for both surveillance and case management. The recently described method (15,20) of targeted amplicon deep sequencing (TADS) of individual samples overcomes these limitations. While each method has its advantages, TADS is best suited for assessing the allele frequencies of specific genes at the individual patient level in a population.…”

Overlap Extension Barcoding for the Next Generation Sequencing and Genotyping of Plasmodium falciparum in Individual Patients in Western Kenya

“…Population genetics offers a research toolset to elucidate the parasite’s transmission patterns and define and track medically-important genotypes such as those responsible for more severe disease or conferring drug- or vaccine-resistance345. Recent advances in next generation sequencing (NGS) technology have broadened the utility of these approaches6, allowing more comprehensive detection of parasite genotypes present in complex parasitemias7. In doing so, these approaches can enable better understanding of minority variants including their transmission8, contribution to parasite population structure, and involvement in drug and vaccine candidate resistance9.…”