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mRNA translation plays a critical role in determining proteome composition. In health, regulation of mRNA translation facilitates rapid gene expression responses to intra- and extracellular signals. Moreover, dysregulated mRNA translation is a common feature in disease states, including neurological disorders and cancer. Yet, most studies of gene expression focus on analysis of mRNA levels, leaving variations in translational efficiencies largely uncharacterized. Here, we outline procedures to identify mRNA-selective alterations in translational efficiencies on a transcriptome-wide scale using the anota2seq package. Anota2seq compares expression data originating from translated mRNA to data from matched total mRNA to identify changes in translated mRNA not paralleled by corresponding changes in total mRNA (interpreted as changes in translational efficiencies impacting protein levels), congruent changes in …