Fig. 2

ORFik functionality. a ORFik supports a number of preprocessing steps including (1) removal of rRNA contamination) and other ncRNA), (2) Alignment to genome or transcriptome, (3) selecting 80S read lengths that display 3 nt periodicity characteristic of ribosome translocation over translated regions (see Additional file 1: Fig. S1). b To identify the P-sites for reads (top illustration) from an 80S library ORFik performs change point detection on the 5′ ends of reads across a window over translation initiation sites. This determines the location of reads from initiating ribosomes and the distance from their 5′ end to the start codon. This is done separately for each read length (heatmap). c Examples of figures used to perform quality control on the data from a Ribo-seq experiment with four samples. Left column: The percentage of reads in each translation frame over CDSs after P-shifting, stratified by read-length. Right column: (1) Number of aligned reads after filtering contaminants. (2) Percentage of reads aligning to various transcript types (> 1%). (3) Percentage alignment of each transcript type in the "Other" group. (4) Percentage of reads aligning to mRNA falling into the CDS or UTRs. For more QC examples see Additional file 1: Figs. S5 and S6. d Read coverage tracks for scanning ribosomal small subunit (SSU), 80S translating ribosomes and CAGE-defined transcription start sites (TSS) for a model gene. The gene has two transcript isoforms with different TSSs and 5′UTRs. The second isoform harbors an additional uORF leading to translational repression through ribosome dissociation (uORF 1). ORFik can assist in detecting such differences in isoform through differential expression, visualization, and metrics