Differential analysis of translation efficiency and usage of open reading frames using DOTSeq

Protein synthesis is a key cellular process in which mRNAs are translated into proteins by ribosomes. This process is tightly regulated, enabling cells to control protein output in response to specific cellular states. Ribosome profiling captures translatomic landscapes across conditions, but existing computational tools for differential translation analysis operate at the gene level, overlooking translational control at the level of multiple open reading frames (ORFs). Here, we present DOTSeq , a Differential ORF Translation statistical framework that enables systematic discovery of translational control events within genes. DOTSeq offers differential analyses of ORF usage and translation efficiency across biological conditions. These modules allow global detection of cis -regulatory events, such as upstream ORF (uORF)-mediated translational control. Benchmarking on simulated datasets demonstrates DOTSeq ‘s sensitivity to subtle regulatory signals, particularly in modest effect sizes where most biological signals occur and where existing tools often show limited sensitivity. DOTSeq provides a flexible and powerful approach for dissecting the complexity of translational control.