Fig. 1

Summary of a Ribo-seq experiment and subsequent computational analysis. a The experiment starts with isolation of mRNA ribosome complexes, followed by nuclease digestion of mRNA sequences that are not protected by associated ribosomes. Purification of the mRNA fragments shielded by the ribosomes is then carried out, followed by library generation, deep sequencing, and data analysis. b Plot of ribosome protected fragment counts along the translation start and end sites. During the process of translation, the p-site holds the amino acid that is linked with the growing polypeptide chain, and therefore more accurately represents the exact position or codon within the coding sequence that the ribosome is interacting with. Because of this, an adjustment must be made to account for the positional differences between the first position of a read and the corresponding p-site of the read. Pausing of ribosomes at each codon leads to trinucleotide periodicity. The majority of reads are expected to be ‘in frame’ with the start codon. c Differences in Ribo-seq and RNA-seq read densities caused around the start and stop codons. In contrast to RNA-seq data, Ribo-seq data tends to show a large peak around the start codon, as well as larger percentages of sequencing reads in frame with the start codon