Additional file 9 of Multiple links between 5-methylcytosine content of mRNA and translation

Figure S4. m5C candidate site call reproducibility across biological replicates and effects of non-conversion ‘noise suppression’. Related to Fig. 2. A: Pair-wise scatter plot comparisons of transcriptome-wide candidate sites called in composite libraries of each biological replicate. Sites shown passed the 80CT, 30RC and 5C filter in their respective composite library (a non-conversion cut-off was not applied). Further to that, only sites with coverage in all three replicates were used. The adjusted R-squared value following linear regression is shown. B: Effect of the 3C and S/N90 filters on candidate site calling in different RNA types. The number of candidate sites that passed the 80CT, 30RC, 5C filter in their respective composite library and fulfilled the 10MM criterion are listed. C: Position of candidate sites in the tRNA cloverleaf consensus structure. Each circle indicates a nucleotide position within the tRNA cloverleaf structure, with blue filled circles indicating position at which candidate sites were identified. Iso-acceptors found to carry the candidate site are identified by the single letter amino acid code. D: Genetic code table highlighting tRNA iso-decoders with candidate sites in blue. C-D: Of note, we detected the NSUN2-dependent sites at the edge of the variable loop at position C48–50 in a variety of tRNA iso-decoders, as well as at position C34 of intron-containing tRNALeu (CAA). We further identified the TRDMT1-dependent modification of C38 in tRNAAsp (GUC). Interestingly, we also detected several candidate sites at structural position C72. The established NSUN6-dependent sites in tRNAThr (UGU) and tRNACys (GCA) iso-decoders did not receive read coverage. Instead, we saw clear non-conversion at C72 in tRNAIle (UAU), tRNALys (CUU) and tRNASer (ACU); these might be novel NSUN6 substrates. C70 in tRNAGly (CCC) is indicated in the figure, however, detection of this site is heavily driven by the terminal base of reads in one direction, thus likely suspect.