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. 2020 May 12;9:e51015. doi: 10.7554/eLife.51015

Figure 5. Competition growth assays of pfk13 mutant and wild-type parasites.

(a) Frequency of wild-type and mutant parasites in co-culture, as measured by TaqMan allelic discrimination qPCR. Data show the percentage of pfk13 mutant parasites in the culture over 60 days with sampling every two days. Error bars represent the SEM of pfk13 mutant allele frequency between the two biological replicates (including two technical replicates for qPCR). A percentage below 50% indicates the mutant was less fit than the isogenic pfk13 wild-type line. (b) Percentage change per generation of pfk13 mutant allele frequency relative to wild-type. Data show that pfk13 mutations confer an in vitro fitness cost in both parasite lines. Differences in growth rates were calculated as the percent change in pfk13 mutant allele frequency averaged over 30 generations. Error bars represent the SEM of percentage growth change between the two biological sampling experiments calculated for every generation in each co-culture. Significance was calculated using the Wilcoxon signed-rank test in every generation across the two biological replicate experiments. **p<0.01, ***p<0.001; ns: not significant.

Figure 5—source data 1. Proportion of pfk13 mutants compared to pfk13 wild-type during 60 days of in vitro co-culture.

Figure 5.

Figure 5—figure supplement 1. Representative standard curves for qPCR reactions targeting pfk13 C580/C580Y or pfk13 R539/R539T allele.

Figure 5—figure supplement 1.

(a, b) Standard curves showing good amplification efficiency (between 88% and 95%) and high sensitivity using 10-fold serially diluted genomic DNA obtained from wild-type pfk13 C580 and R539 or mutant pfk13 C580Y or R539T parasites. (c, d) Scatter plots for percent wild-type and mutant alleles in multiplexed qPCR assays using pre-defined mixtures of plasmids. We used mixtures comprising of pfk13 C580 and C580Y or R539 and R539T expressing plasmids in fixed molar ratios of wild-type: mutant alleles (0:100, 20:80, 40:60, 50:50, 60:40, 80:20, 100:0) to validate the specificity of using TaqMan qPCR assays to determine the pfk13 allele frequency.
Figure 5—figure supplement 1—source data 1. Data for establishment of standard curves and pre-defined set of mixtures of each pfk13 allele (C580Y or R539T) used in the optimization of the Taqman allelic discrimination qPCR assays.
Figure 5—figure supplement 2. Reproducibility of TaqMan allelic discrimination qPCR performed on R086R539T and R086 parasites.

Figure 5—figure supplement 2.

(a) Scatter plots show the percentage of pfk13 R539T parasites in two separate qPCR technical replicate runs which correlate perfectly. (b) Scatter plots show the percentage of pfk13 R539T parasites in two independent sampling replicates over 60 days in culture, which showed consistent trends.
Figure 5—figure supplement 2—source data 1. Data for the reproducibility of the Taqman allelic discrimination assay across qPCR repeats and sampling replicates performed with R086WT vs R086R539T parasites.