Analysis of mRNA-seq and Ribo-seq measurements

The ribosomal profiling (or Ribo-seq) is a method that gives quantitative information of ribosome footprints in a single nucleotide resolution [20]. Ribo-seq/mRNA-seq raw data was obtained the from the NCBI GEO database [16] (accession {"type":"entrez-geo","attrs":{"text":"GSE34082","term_id":"34082"}}GSE34082). Transcript sequences were obtained from EnsEMBL for S. cerevisiae (R64-1-1, Ensembl release 78). We trimmed 3’poly-A adaptors from the reads using Cutadapt, version 1.8.3 [42]. Following, we utilized Bowtie [43] to map them to the S. cerevisiae transcriptome (version 1.1.1). In the first phase (for Ribo-seq reads only), we discarded reads that mapped to rRNA and tRNA sequences (Bowtie parameters ‘–n 2 –seedlen 23 –k 1 --norc’). In the second phase (for both Ribo-seq and mRNA-seq reads), we mapped the remaining reads to the transcriptome (Bowtie parameters ‘–v 2 –a --strata --best --norc –m 200’). We tried to extend alignments to their maximal length by comparing the poly-A adaptor with the aligned transcript until reaching the maximal allowed error (i.e. two mismatches across the read, with 3'end mismatches avoided). We filtered out reads longer than 32 nt or shorter than 23 nt for Ribo-seq reads, and filtered out reads longer than 40 nt or shorter than 25 nt for mRNA-seq reads. Unique alignments were first assigned to the RNA/ribosome occupancy profiles. For multiple alignments, the best alignments in terms of number of mismatches were kept. Then, multiple aligned reads were distributed between locations according to the distribution of unique ribosomal/RNA reads in the respective surrounding regions. To this end, a 100 nt window was used to compute the read count density RCD _i (total read counts in the window divided by length, based on unique reads) in vicinity of the M multiple aligned positions in the transcriptome, and the fraction of a read assigned to each position was determined as: RCDi/∑j=1MRCDj. For ribosome footprints, the location of the A-site was set 15 nt downstream of the 5' of the read.