ORF Relative Branch Length (ORBL) measures cross-species evolutionary conservation and constraint on the "ORFness" of an open reading frame (ORF), without regard to conservation of the encoded amino acid sequence. It is intended to detect ORFs encoding poorly conserved peptides, as well as ORFs whose translation is functional but which do not necessarily encode a functional peptide, such as regulatory uORFs. ORBL uses multispecies whole genome alignments to obtain the local alignment of the ORF in a particular clade, and then computes two scores, ORBLv and ORBLq. ORBLv measures conservation of ORFness by calculating the relative branch length of the phylogenetic tree of species in the alignment in which there is an aligned ATG start codon, stop codon, and open reading frame. ORBLq measures evolutionary constraint on ORFness by calculating the quantile of its ORBLv score among the ORBLv scores of untranslated ORFs of the same biotype and similar length, in order to correct for conservation due to chance or to constraint on an overlapping coding sequence (CDS). By detecting evolutionary constraint on translation itself, ORBL expands the scope of comparative genomics to detect functional ORFs that would be missed by conventional protein conservation analyses.

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