Characterizing temporal variability and repeatability of dose-dependent functional genomics approach for evaluating triclosan toxification

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Dose-dependent functional genomics approach has shown great advantage in identifying the molecular initiating event (MIE) of chemical toxification and yielding point of departure (POD) at genome-wide scale. However, POD variability and repeatability derived from experimental design (settings of dose, replicate number, and exposure time) has not been fully determined. In this work, we evaluated POD profiles perturbed by triclosan (TCS) using dose-dependent functional genomics approach in Saccharomyces cerevisiae at multiple time points (9 h, 24 h and 48 h). The full dataset (total 9 concentrations with 6 replicates per treatment) at 9 h was subsampled 484 times to generate subsets of 4 dose groups (Dose A - Dose D with varied concentration range and spacing) and 5 replicate numbers (2 reps - 6 reps). Firstly, given the accuracy of POD and the experimental cost, the POD profiles from 484 subsampled datasets demonstrated that the Dose C group (space narrow at high concentrations and wide dose range) with three replicates was best choice at both gene and pathway levels. Secondly, the variability of POD was found to be relatively robustness and stability across different experimental designs, but POD was more dependent on the dose range and interval than the number of replicates. Thirdly, MIE of TCS toxification was identified to be the glycerophospholipid metabolism pathway at all-time points, supporting the ability of our approach to accurately recognize MIE of chemical toxification at both short- and long-term exposure. Finally, we identified and validated 13 key mutant strains involved in MIE of TCS toxification, which could serve as biomarkers for TCS exposure. Taken together, our work evaluated the repeatability of dose-dependent functional genomics approach and the variability of POD and MIE of TCS toxification, which will benefit the experimental design for future dose-dependent functional genomics study.