Extended Data Fig. 9: Reproduciblity of viral ORFs translation.

a–c, Scatter plot presenting the correlation of footprint densities at each initiation site relative to a 8bp window 3 nucleotides downstream of the initiation site in LTM treated samples between two biological replicates of infected Vero E6, 5 hpi (a), between two biological replicates of infected Calu3 cells, 7 hpi (b) and between infected Vero E6 cells at 5 hpi and Calu3 cells at 7 hpi (c). Canonical ORFs are marked in dark green and newly identified ORFs are marked in light green. As a control, the relative occupancy of random positions was calculated in the same way (grey). Dashed lines mark the equal ratio of one. Spearman’s R is presented. d, The position of ribosome footprints relative to the reading frame in all canonical ORFs and novel ORFs excluding in-frame iORFs are presented for our measurements of infected Vero E6 cells at 5 hpi (lower panels) and infected Calu3 cells (upper panels). Filled and open rectangles indicate the canonical and novel ORFs, respectively. ORFs starting in near cognate start codons are labelled with stripes. The frame of the footprints is summed on each of the indicated regions and is presented relative to the frame of the canonical ORF in each of these loci. In all non-overlapping regions, beside ORF10 in Vero E6 cells, clear enrichment to the translated frame is observed indicating active translation. For out-of-frame overlapping ORFs the bar of its frame is labelled by colour and the percentage of the footprints that originate from the overlapping frame as was calculated from linear regression is presented together with the corresponding P value for the contribution of the out-of-frame ORF to the frame distribution of the total reads in this region, using two degrees of freedom that reflect the two replicates. In two out-of-frame overlapping ORFs, ORF3.iORF2 and ORF8.iORF the expression relative to the main ORF was low and did not lead to a significant shift in the translation signal. In all other ORFs there is a significant signal in the alternative frame indicating active translation.