Fig. 1

Measurement intrahost variant frequencies are more accurate at high frequencies and are susceptible to input concentrations and coverage depths. a We created genetically diverse virus populations by mixing two Zika virus isolates with 159 consensus nucleotide differences to test the effects of PCR amplification prior to sequencing to measure intrahost single-nucleotide variant (iSNV) frequencies. For these initial experiments, we amplified three ~ 400 bp regions of the Zika virus genome using primers without any mismatches to either of the mixed virus (shown as amplicons 5, 24, and 33). "Amplicon 5" contains 5 iSNV sites, "amplicon 24" contains 8 iSNV sites, and "amplicon 33" contains 5 iSNV sites. b We created virus populations containing 50%, 25%, 14%, 7%, 3%, 1.5%, and 0.8% virus #2 to test the impact of PCR amplification prior to sequencing on measuring ranges of iSNV frequencies. The data points represent individual iSNVs amplified and sequenced in triplicate from each population (colored by amplicon 5, 24, or 33 as shown in a. c We 10-fold serially diluted a mixed population containing 14% of virus #2 (expected, dotted line) from 100,000 to 10 copies to test the effects of input concentrations on accurate iSNV measurements. d We randomly downsampled the datasets generated from 1000 input virus RNA copies containing 3% virus #2 to set coverage depths (sequenced nucleotides [nt] per genome position) to determine the minimum coverage needed to yield accurate iSNV measurements. For c and d, the Levene’s test was used to assess equality among variances of iSNV measurements from each coverage depth (ns, not significant; *, p < 0.05). Data shown as means with standard deviations