Rather than discard all transcripts with large ORFs, as we did here, one option to discriminate between transcripts that are coding versus noncoding is to analyze the frequency of synonymous codon substitutions (PhyloCSF) [40]. However, this approach is limited to ORFs that can be aligned across species, potentially missing recently evolved or otherwise nonconserved novel protein coding genes. Importantly, our approach of removing all transcripts with large open reading frames effectively removed transcripts with significant predicted coding potential (Figure 2C), indicating that using an ORF size cutoff is at least as conservative as filtering based on PhyloCSF analysis. The lack of engagement of the ribosome, observed with ribosomal profiling data, confirms the stringency of the ORF cutoff filter (Figure 2B). Further analysis of these removed large ORF-containing intergenic transcripts is outside the scope of this study, but we have included these annotations for investigators interested in further analyzing their coding potential in search of novel protein coding genes (Dataset S10).
