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By capturing and sequencing the RNA fragments protected by translating ribosomes, ribosome profiling provides snapshots of translation at subcodon resolution. The growing needs for comprehensive annotation and characterization of the context-dependent translatomes are calling for an efficient and unbiased method to accurately recover the signal of active translation from the ribosome profiling data. Here we present our new method, RiboCode, for such purpose. Being tested with simulated and real ribosome profiling data, and validated with cell type-specific QTI-seq and mass spectrometry data, RiboCode exhibits superior efficiency, sensitivity, and accuracy for de novo annotation of the translatome, which covers various types of ORFs in the previously annotated coding and non-coding regions. As an example, RiboCode was applied to assemble the context-specific translatomes of yeast under normal and stress conditions. Comparisons among these translatomes revealed stress-activated novel upstream and downstream ORFs, some of which are associated with translational dysregulations of the annotated main ORFs under the stress conditions.

De novo annotation and characterization of the translatome with ribosome profiling data