Ribo-seq count table for all genes analyzed.

Acute cellular stress is known to induce a global reduction in mRNA translation through suppression of cap dependent translation. Selective translation in response to acute stress has been shown to play important roles in regulating the stress response. However, accurately profiling translational changes transcriptome-wide in response to acute cellular stress has been challenging. Commonly used data normalization methods operate on the assumption that any systematic shifts are experimental artifacts. Consequently, if applied to profiling acute cellular stress-induced mRNA translation changes, these methods are expected to produce biased estimates. To address this issue, we designed, produced, and evaluated a panel of 16 oligomers to serve as external standards for ribosome profiling studies. Using Sodium Arsenite treatment-induced oxidative stress in lymphoblastoid cell lines as a model system, we applied spike-in oligomers as external standards. We found our spike-in oligomers to display a strong linear correlation between the observed and the expected quantification, with small ratio compression at the lower concentration range. Using the expected fold changes constructed from spike-in controls, we found in our dataset that TMM normalization, a popular global scaling normalization approach, produced 87.5% false positives at a significant cutoff that is expected to produce only 10% false positive discoveries. In addition, TMM normalization produced a systematic shift of fold change by 3.25 fold. These results highlight the consequences of applying global scaling approaches to conditions that clearly violate their key assumptions. In contrast, we found RUVg normalization using spike-in oligomers as control genes recapitulated the expected stress induced global reduction of translation and resulted in little, if any, systematic shifts in the expected fold change. Our results clearly demonstrated the utility of our spike-in oligomers, both for constructing expected results as controls and for data normalization.