DENetwork unveils non-differentially expressed genes with functional relevance across conditions through information flow perturbation.

Differential gene expression (DE) analysis of RNA-sequencing (RNA-seq) data is a standard approach for identifying phenotypic differences between conditions. However, traditional DE methods such as DESeq2 focus on expression changes alone, often overlooking non-differentially expressed (non-DE) genes that may play key regulatory roles. This limits their ability to identify upstream drivers of transcriptomic variation. To address this gap, we introduce DENetwork, a network-based approach that prioritizes genes based on their influence on global information flow. Each gene is scored using an in silico knockout strategy that quantifies its impact across the inferred gene network, capturing both DE and non-DE genes with potential functional relevance. DENetwork deciphers intricate regulatory and signaling networks driving transcriptomic variations between conditions with distinct phenotypes. Across simulated and disease-relevant RNA-seq datasets, DENetwork identifies non-DE regulators enriched in known pathways and phenotypic associations, providing mechanistic insights missed by standard DE analysis, with implications for target discovery and intervention.