Fig. 2

Measures of intrahost variant frequencies are sensitive to primer mismatches. a To assess the impacts of primer mismatches on accurately measuring intrahost single-nucleotide variants (iSNVs), we sequenced a mixed Zika virus population using 35 overlapping PCR amplicons (see “Amplicon scheme” above panel). The virus population contained 10% virus #2 (Expected) and 1000 virus RNA copies were amplified and sequenced in triplicate. The amplicons and iSNVs are colored according to the number of mismatches in the primer sequences used to generate that amplicon. Data shown as means and ranges. b To account for unequal iSNV sites within each amplicon, the iSNV frequencies on each amplicon were averaged to produce a haplotype frequency for virus #2 mixed at 10% (Expected). Data shown as means and ranges. c We calculated the deviations between the measured and expected virus #2 haplotype frequencies (absolute value of the log₂ fold change) to assess the bias introduced during PCR of amplicons containing primer mismatches to virus #2 (*, Welch’s t test, p < 0.05). Data shown as means and standard deviations. d We plotted the deviations from expected haplotype frequencies by the distance of mismatches from the 3′ end of the primer to investigate the impact of mismatch location. If more than one mismatch was present on a primer pair (orange), the data is shown using the closest mismatch to the 3′ end. Mismatches closer to the 3′ end of the primer are more likely decrease the accuracy of iSNV or haplotype measurements from that amplicon (correlation by Pearson r, p < 0.05). Data shown as the mean from all three replicates