Extended Data Fig. 5: Analysis of per-codon ribosome density.

5′ normalized ribosome-protected mRNA fragment (RPF) density for a, all codons and b, the top 3 slowest/fastest codons. Plotting the normalized density of Ribo-seq read 5′ ends relative to each codon/read length/sample shows two strongly variable regions corresponding to 5′- and 3′-end cut site biases during nuclease digestion. A third variable region in between corresponds to RPFs with their A/P-sites positioned over the codon. We infer the location of the A-site as the 3 bp region showing the most inter-codon variability (see Methods), and use the normalized occupancy here to measure codon density, and variance between codons. Independently, this region also identifies the location of intra-codon variability between samples. c, Per-codon correlation between tRNA availability calculated from tRNA qPCR array (see Methods), and the ribosome occupancy of that codon when positioned in the A-site of the ribosome footprint. Center is maximum likelihood slope, ribbon is 95% CI. d, Correlation between ribosome occupancy per codon and the optimality of the codon as defined in ref. ⁴⁶, with the mean across all stages shown. Association between paired samples in (c, d) was tested with Pearson’s product moment correlation coefficient. Associated with Fig. 5. See also Supplementary Table 4.