Group Heteroscedasticity - A Silent Saboteur of Power and False Discovery in RNA-Seq Differential Expression

Chatterjee, S.; Fadikar, A.; Hanumesh, V.; Meshram, S. S.; Zoh, R. S.; Ma, S.; Arunkumar, G.; Mallick, H.

doi:10.1101/2024.04.01.587633

Home/Archive/bioRxiv

bioRxiv · 10.1101/2024.04.01.587633

Group Heteroscedasticity - A Silent Saboteur of Power and False Discovery in RNA-Seq Differential Expression

Chatterjee, S.·Fadikar, A.·Hanumesh, V.·Meshram, S. S.·Zoh, R. S.·Ma, S.·Arunkumar, G.·Mallick, H.

View original source ↗Export metadata ↓

Abstract

Despite the availability of several high-profile, state-of-the-art methods, analyzing bulk RNA-Seq data continues to face significant challenges. Evidence from recent studies has highlighted that popular differential expression (DE) tools, such as edgeR and DESeq2, are susceptible to an alarmingly high false discovery rate (FDR). These studies suggest that the FDR inflation observed in these models could be attributed to issues such as violations of parametric assumptions or an inability to effectively handle outliers in the data. Here, we argue that group heteroscedasticity can also contribute to this elevated FDR, a phenomenon largely overlooked by the research community. We introduce a novel statistical model, Robseq, designed for effective per-feature modeling in differential analysis, particularly when the assumption of group homoscedasticity is unmet. Robseq utilizes well-established statistical machinery from the robust statistics literature, including M-estimators to robustly estimate gene expression level changes and Huber-Cameron variance estimators to calculate robust standard errors in heteroscedastic settings. Additionally, it incorporates a degrees of freedom adjustment for the Welch t-statistic, based on Bell-McCaffreys recommendation, for inferential purposes, effectively addressing the problem of FDR inflation in RNA-Seq differential expression. Through detailed simulations and comprehensive benchmarking, we show that Robseq successfully maintains the false discovery and type-I error rates at nominal levels while retaining high statistical power compared to well-known DE methods. Analysis of population-level RNA-Seq data further demonstrates that Robseq is capable of identifying biologically significant signals and pathways implicated in complex human diseases that otherwise cannot be revealed by published methods. The implementation of Robseq is publicly available as an R package at https://github.com/schatterjee30/Robseq.

Explore related subjects

bioinformatics Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Chatterjee, S., Fadikar, A., Hanumesh, V., Meshram, S. S., Zoh, R. S., Ma, S., Arunkumar, G., Mallick, H.. 2024-04-02. Group Heteroscedasticity - A Silent Saboteur of Power and False Discovery in RNA-Seq Differential Expression. https://doi.org/10.1101/2024.04.01.587633

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

spatialMET: an open and scalable framework for spatial metabolomics analysis

Mass spectrometry imaging (MSI) enables spatially resolved metabolomics in intact tissue sections, but analysis remains challenging at scale. Existing MSI workflows often require users to combine multiple software tools, while others rely on proprietary vendor software that limits interoperability and reproducibility. To address these challenges, we developed spatialMET, an open-source framework that provides an end-to-end workflow for MSI analysis. spatialMET provides a unified platform for preprocessing, spatial domain detection, and visualization. Downstream analyses include differential abundance testing, spatial autocorrelation and gradient analysis, dimensionality reduction, and correlation network analysis. Spatial domain detection uses hcdist, a C-based hierarchical clustering implementation that substantially reduces runtime and memory use relative to existing R-based approaches. spatialMET can be run through an interactive R Shiny application or as a standalone command-line workflow for larger datasets or high-performance computing environments. Applied to mouse small cell lung cancer MALDI-MSI data containing 284,673 pixels, spatialMET identified tumor-associated, stromal, and adjacent lung spatial domains that aligned with matched histology. Differential abundance analysis identified 117 m/z features that differed between tumor and stromal regions, while spatial autocorrelation analyses revealed spatially structured abundance patterns. Applying spatialMET to mouse lung adenocarcinoma data from an entire lung lobe containing 338,477 pixels further demonstrated scalability and captured spatial heterogeneity across tumor and surrounding lung tissue. In summary, spatialMET provides a scalable, open-source framework for end-to-end spatial metabolomics analysis, and it is distributed as a Docker container for reproducible deployment. Source code and installation instructions are available at https://github.com/biodatalab/spatialMET.

bioinformatics↗

bioRxiv2026

Probing the transcriptome response to shivering in skeletal muscle using a multilayered bioinformatics approach

Cold acclimation holds therapeutic potential for improving metabolic health. We previously demonstrated that repeated cold-induced shivering enhances insulin sensitivity in humans. However, the molecular pathways that underlie the skeletal muscle shivering response, and how these relate to beneficial physiological effects, remain poorly understood. In this study, we combined complementary bioinformatics approaches to allow in-depth analysis of the transcriptomic response of human skeletal muscle to repeated shivering. We identified a robust transcriptional signature and show a sex-specific component in the shivering skeletal muscle response, which seemed to diminish following cold adaptation. Our findings provide mechanistic insights into cold-induced muscle adaptations, shed light on potential interesting molecular targets for further investigation, and emphasize the importance of including both sexes in future cold acclimation studies.

bioinformatics↗

bioRxiv2026

An Information Geometry approach to model topological trajectories and Gene Expression Radius from UMAP geometry.

Understanding the relationship between gene expression dynamics and cellular identity remains a central challenge in single cell biology. Here, we introduce a novel computational and mathematical framework that integrates information geometry, fuzzy topology, and UMAP analysis to model gene expression landscapes derived from single cell RNA sequencing data. We formalize gene expression data as a fuzzy topological space, where interactions between expression points are governed by probabilistic distributions inspired by manifold learning approaches such as UMAP. Within this framework, we define an information geometric structure through a Fisher metric induced by these distributions, enabling the computation of geodesic trajectories that capture cellular differentiation processes. A key contribution of this work is the derivation of analytical conditions, expressed as expression radius formulas, that characterize local neighborhoods in gene expression space. These conditions allow for the identification of genes associated with stem cell states and predictions in transitional cell types in future work. Application of the proposed framework to single cell datasets reveals biologically meaningful gene sets enriched in key regulatory pathways and transcription factors, demonstrating the capacity of our approach to uncover latent structure in complex gene expression data. Our results suggest that integrating differential geometry with statistical learning theory offers a powerful paradigm for modeling genotype and phenotype relationships and cellular state transitions, with potential implications for precision medicine and systems biology.

bioinformatics↗