Multimapping confounds ribosome profiling analysis: A case‐study of the Hsp90 molecular chaperone

Ribosome profiling (Ribo‐seq) can potentially provide detailed information about ribosome position on transcripts and estimates of protein translation levels in vivo . Hsp90 chaperones, which play a critical role in stress tolerance, have characteristic patterns of differential expression under nonstressed and heat shock conditions. By analyzing published Ribo‐seq data for the Hsp90 chaperones in S. cerevisiae , we find wide‐ranging artifacts originating from “multimapping” reads (reads that cannot be uniquely assigned to one position), which constitute ~25% of typical S. cerevisiae Ribo‐seq datasets and ~80% of the reads from HEK293 cells. Estimates of Hsp90 protein production as determined by Ribo‐seq are reproducible but not robust, with inferred expression levels that can change 10‐fold depending on how multimapping reads are processed. The differential expression of Hsp90 chaperones under nonstressed and heat shock conditions creates artificial peaks and valleys in their ribosome profiles that give a false impression of regulated translational pausing. Indeed, we find that multimapping can even create an appearance of reproducibility to the shape of the Hsp90 ribosome profiles from biological replicates. Adding further complexity, this artificial reproducibility is dependent on the computational method used to construct the ribosome profile. Given the ubiquity of multimapping reads in Ribo‐seq experiments and the complexity of artifacts associated with multimapping, we developed a publicly available computational tool to identify transcripts most at risk for multimapping artifacts. In doing so, we identify biological pathways that are enriched in multimapping transcripts, meaning that particular biological pathways will be highly susceptible to multimapping artifacts.