De Novo Annotation And Characterization Of The Translatome With Ribosome Profiling Data

By capturing and sequencing the RNA fragments protected by translating ribosomes, ribosome profiling sketches the landscape of translation at subcodon resolution. We developed a new method, RiboCode, which uses ribosome profiling data to assess the translation of each RNA transcript genome-wide. As shown by multiple tests with simulated data and 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 novel ORFs in the previously annotated coding and non-coding regions and overlapping ORFs. Finally, to showcase its application, we applied RiboCode on a published ribosome profiling dataset and assembled the context-dependent translatomes of yeast under normal condition, heat shock, and oxidative stress. Comparisons among these translatomes revealed stress-activated novel upstream and downstream ORFs, some of which are associated with potential translational dysregulations of the main protein coding ORFs in response to the stress signals.