Additional file 18 of Multiple links between 5-methylcytosine content of mRNA and translation

Figure S10. Relationship between non-conversion level and mRNA translation state for candidate m5C sites with insufficient coverage in bsRNA-seq fraction 1. Related to Fig. 5. bsRNA-seq libraries were grouped as biological triplicates per fraction (e.g. LibB 1, LibC1 and LibE1 each report on sites detected in bsRNA-seq fraction 1), allowing the calculation of average non-conversion levels per individual site and per fraction. A: Non-conversion levels per individual site across the polysome profile were partitioned into nine soft clusters (C1–9 arranged here by similarity in trend with polysome profile) using Mfuzz. 315 candidate m5C sites were included based on having coverage in at least 9 out of 12 bsRNA-seq fraction samples and ≥ 10 average coverage in bsRNA-seq fractions 2–4 but failing this criterion for fraction 1. Degree of cluster ‘membership’ is indicated by the colour scale depicted to the right of panel A. Candidate sites with high membership (blue) have the best match to the respective cluster’s overall pattern. The legend to the right of panel A gives a colour/significance key applicable to all panels. Insets are: pie charts showing distribution of cluster members across different mRNA regions; boxplots showing site non-conversion distribution of cluster members across the polysome profile. Asterisks indicate significance p-value from unpaired, two-tailed Student’s t-test comparing the means of adjacent fractions. B: Mfuzz clusters from panel A were grouped into three translation state trend categories by visual inspection, showing a negative (clusters C3,4,6,9; N = 133), neutral (clusters C2,8,1; N = 75) or positive trend (clusters C5,7; N = 107) with polysome association. Top panels: line graphs displaying individual site average non-conversion levels across fractions. Middle panels: boxplots showing distribution of site non-conversion levels in each fraction. Asterisks indicate significance p-value from unpaired, two-tailed Student’s t-test comparing the means of adjacent fractions. Bottom panels: boxplots showing distribution of site coverage in each fraction. C: Stacked bar charts showing distribution of sites in the different translation state trend categories across mRNA regions (top) and distribution of sites in different mRNA regions across translation state trend categories (bottom). Asterisks indicate significance p-values following binomial test against the distribution of all sites. The legend to the right of panel A gives a colour/significance key applicable to all panels.