Comparative analysis of targeted next-generation sequencing for Plasmodium falciparum drug resistance markers

Well-defined molecular resistance markers are available for a range of antimalarial drugs, and molecular surveillance is increasingly important for monitoring antimalarial drug resistance. Different genotyping platforms are available, but these have not been compared in detail. We compared Targeted Amplicon Deep sequencing (TADs) using Ion Torrent PGM with Illumina MiSeq for the typing of antimalarial drug resistance genes. We developed and validated protocols to type the molecular resistance markers pfcrt, pfdhfr, pfdhps, pfmdr1, pfkelch, and pfcytochrome b, in Plasmodium falciparum for the Ion Torrent PGM and Illumina MiSeq sequencing platforms. With P. falciparum 3D7 and K1 as reference strains, whole blood samples (N = 20) and blood spots from Rapid Diagnostic Test (RDT) samples (N = 5) from patients with uncomplicated falciparum malaria from Ubon Ratchathani were assessed on both platforms and compared for coverage (average reads per amplicon), sequencing accuracy, variant accuracy, false positive rate, false negative rate, and alternative allele detection, with conventional Sanger sequencing as the reference method for SNP calling. Both whole blood and RDT samples could be successfully sequenced using the Ion Torrent PGM and Illumina MiSeq platforms. Coverage of reads per amplicon was higher with Illumina MiSeq (28,886 reads) than with Ion Torrent PGM (1754 reads). In laboratory generated artificial mixed infections, the two platforms could detect the minor allele down to 1% density at 500X coverage. SNPs calls from both platforms were in complete agreement with conventional Sanger sequencing. The methods can be multiplexed with up to 96 samples per run, which reduces cost by 86% compared to conventional Sanger sequencing. Both platforms, using the developed TAD protocols, provide an accurate method for molecular surveillance of drug resistance markers in P. falciparum, but Illumina MiSeq provides higher coverage than Ion Torrent PGM.