Fig. 2

Detection of translation in annotated and non-annotated ORFs. a Pipeline used in this study. An ORF was considered translated, in each stage, if it showed transcription (RPKM > 1 in one of two RNA-Seq replicates), reproducible ribosomal binding (RPKM > 1 in both Poly-Ribo-Seq replicates) and a significant framing of FPs (p value < 0.01 in the binomial test for framed-reads). b Metagene plot for codon framing of 32-nt ribosome footprints across all ORFs analyzed in this study, showing three-nucleotide periodicity (framing) in the third position of each codon (frame 2, blue). Numbers on top denote the distances between the 5′-end of ribosomal footprints and START/STOP codons. c The observed framing for 32-nt ribosome footprints is consistent across embryonic stages (combined replicates per stage). d Logic of per-ORF analysis of framing probabilities for each ORF at a given stage using the binomial test. e Density of 32-nt genome-aligned Poly-Ribo-Seq reads for all experiments, per frame, and corresponding translation probability for the scl-A ORF, as measured by the binomial test (see Table S3B for details). f Numbers of developmentally transcribed, ribosome-bound, and translated canonical ORFs (annotated ORFs with length > 100 codons), short CDSs (annotated ORFs ≤ 100 codons), and uORFs