Comparative Analysis of Plasmodium falciparum Genotyping via SNP Detection, Microsatellite Profiling, and Whole-Genome Sequencing

Abstract

Research efforts to combat antimalarial drug resistance rely on quick, robust, and sensitive methods to genetically characterize Plasmodium falciparum parasites. We developed a single-nucleotide polymorphism (SNP)-based genotyping method that can assess 33 drug resistance-conferring SNPs in dhfr, dhps, pfmdr1, pfcrt, and k13 in nine PCR reactions, performed directly from P. falciparum cultures or infected blood. We also optimized multiplexed fragment analysis and gel electrophoresis-based microsatellite typing methods using a set of five markers that can distinguish 12 laboratory strains of diverse geographical and temporal origin. We demonstrate how these methods can be applied to screen for the multidrug-resistant KEL1/PLA1/PfPailin (KelPP) lineage that has been sweeping across the Greater Mekong Subregion, verify parasite in vitro SNP-editing, identify novel recombinant genetic cross progeny, or cluster strains to infer their geographical origins. Results were compared with Illumina-based whole-genome sequence analysis that provides the most detailed sequence information but is cost-prohibitive. These adaptable, simple, and inexpensive methods can be easily implemented into routine genotyping of P. falciparum parasites in both laboratory and field settings.

Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness

“…To provide a microsatellite (MS) signature for each recombinant progeny, we established a list of MS markers that differentiated RF7 from NF54. For this analysis, we wrote a custom Python script, using the pysam module, to call WGS reads showing insertions or deletions at targeted genomic loci ( 79 ). Integrated Genome Viewer was used to visually verify the presence of MS markers.…”

“…S14). PCRs were set up individually for C2M18, TAA81, BM5, C3M67, TA1, and C13M87, using forward primers fluorescently labeled with 6-FAM (blue), ATTO550/NED (yellow), or ATTO532/VIC (green) as described previously ( 79 ). PCR products treated with ExoSAP-IT Express Reagent (Thermo Fisher Scientific) were sent for capillary electrophoresis with the Liz500 ladder as reference (Genewiz).…”

“…Parasite cultures were monitored for recrudescence by microscopy for up 8 weeks post-electroporation. To screen for successful editing, the k13 and pfcrt loci were amplified directly from parasite cultures using the MyTaq Blood-PCR Kit (Bioline) ( 79 ). PCR products were submitted for Sanger sequencing, and edited parasites were cloned by limiting dilution.…”

“…Each sample was assayed in technical duplicates. The pm2 copy number for each progeny line was calculated by normalizing to the 3D7 control, using the relative quantification method ( 79 ). Primer sequences are listed in table S13.…”

“…Quantitative PCR for determination of pm2 copy number Multiplexed TaqMan quantitative PCR (qPCR) of pm2 labeled with FAM and single-copy β-tubulin labeled with HEX (as an internal control) were performed on gDNAs extracted from the two parents, progeny clones, and gene-edited lines, pre-and postphenotyping. Reactions were run on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific) (79). Five standards of gene fragments, mixed at 1:1, 2:1, 3:1, 4:1, and 5:1 molar ratios of pm2:βtubulin, as well as the 3D7 line (one copy of pm2), were included as copy number controls.…”