Fig. 3: Bioinformatic analysis of the genetic evolution of viral populations obtained by next-generation sequencing.

a Coverage of the next-generation sequencing analysis along the ZIKV genome sequence of viral populations harvested at the indicated time points of the serial passaging of one representative independent run of the evolution experiment. Results are expressed as number of reads per position; schematic representation of ZIKV genome at the top. b, c Time-course quantification of the frequency of the non-parental variants at each position along ZIKV genome in the viral populations harvested in at least one representative independent run of evolution experiment (b) and in the viral populations harvested at passage 6 in three independent runs of evolution experiments (c). In both cases, the frequency of the second most frequent variant is shown. Dotted lines indicate the positions in the viral genome with high standard deviations in several runs of experimental evolution, as defined in Supplementary Fig. 4c. d–f Time-course quantification of the frequency of variants determined by next-generation sequencing. The variants were selected when the standard deviations of their frequencies were: ≥0.1 for all the three independent runs of experimental evolution (n = 3; n referred to one replicate of one condition at given time of harvest) (d); ≥ 0.1 for a minimum of 2 samples (e), and ≥ 0.02 for a minimum of three samples (f), with thresholds defined according to the density of variants relative to their frequency for the pool of all analyzed samples, as presented in Supplementary Fig. 4. The variants are indicated as nucleotide position (e.g., C2340T), the corresponding viral protein (e.g., E) and amino acid change for non-synonymous substitution (e.g., S455L); as also shown on the schematic representation of ZIKV genome organization (a).