Figure 5. Positions within and beyond the canonical promoter are required for efficient replication in the presence of NS2.

The frequency of total non-wild-type nucleotides at each position during minimal replication assays was measured under each condition, and its enrichment, or depletion shown. Similar data were procured for total information content, which considers selection at each individual nucleotide at each individual position (Supplementary Fig. 5) a NS2-dependent selection measured across all of PB1_177:385 against non-wild-type nucleotides, average value across all three replicates provided. Coordinates of deletions from Fig. 4 noted. Coordinates given as the full-length vRNA, regions of functional interest annotated. Data from replication without NS2, and replication with NS2, provided in Supplementary Fig. 7. b Selection on non-wild-type nucleotides in the first 30nt in the vRNA (left) and cRNA (right). Mean and standard deviation graphed. Asterisks indicate regions with a greater-than 2-fold effect size (denoted by red dotted lines) that differ significantly from no effect, one-sample t-test, Benjamini-Hochberg corrected FDR <0.1. The first 20nt of vRNA and cRNA were inferred from 5’ RACE rather than simple amplicon sequencing (Supplementary Fig. 8). Positions further explored in Fig. 6 noted. Inter-replicate correlation plots presented in Supplementary Fig. 9.