ROTS 2.0: A reproducibility-driven framework for robust statistical modeling across diverse high-throughput omics study designs

Reproducibility is fundamental to reliable scientific discoveries. The reproducibility-optimized test statistic (ROTS) is a robust framework designed to identify reproducible features (e.g. genes or proteins) in high-dimensional differential expression analyses such as transcriptomics and proteomics. This is achieved by optimizing the reproducibility of feature rankings under resampling. While originally implemented for univariate settings, ROTS now accommodates multi-group comparisons, survival analysis, linear models, and linear mixed-effects models, broadening its applicability to more complex and clinically relevant experimental designs. Using diverse simulations, benchmark datasets, and real-world case studies, we demonstrate the benefits of ROTS reproducibility optimization compared to the corresponding conventional test statistics. Additionally, we illustrate the utility of the reproducibility characteristics in assessing the overall reliability of the results. To facilitate widespread adoption, ROTS is provided as an open-source software package available through R/Bioconductor. Furthermore, to broaden the user base, we now also provide a Python interface available at pypi.org/project/PyROTS/.