Quantifying Ranking Instability Across Evaluation Protocol Axes in Gene Regulatory Network Benchmarking

Despite theoretical advantages, causal methods for Gene Regulatory Network (GRN) inference from single-cell RNA-seq data consistently fail to match or outperform correlation-based baselines in many realistic benchmarks, a persistent puzzle which casts doubt on the value of causality for this task. We argue that existing benchmarks are insufficiently controlled to answer this question because they evaluate on real or semi-real data where multiple pathologies co-occur, confounding failure modes, and obscuring the specific conditions under which different inference methods excel or fail. To address this gap, we introduce a controlled diagnostic framework that isolates seven biologically motivated pathologies (dropout, latent confounders, cell-type mixing, feedback loops, network density, sample size, and pseudotime drift) and measure how six representative methods spanning three inference paradigms degrade as each pathology intensifies. Across 6,120 controlled experiments, we find that causal methods genuinely dominate in clean and structurally favorable regimes, but specific pathologies (notably dropout and latent confounders) selectively neutralize their advantages. We further introduce an error-type decomposition that reveals methods with similar aggregate accuracy commit qualitatively different errors. To probe whether single-pathology effects persist when multiple stressors co-occur, we perform an interaction sweep over the three most impactful pathologies and find that their joint effects are sub-additive, while also exposing density-conditional cross-overs invisible to single-dial analysis. Our findings offer a nuanced understanding of when and why different methods succeed or fail for GRN inference, providing actionable insights for method development and practical guidance for practitioners.

Inferring gene regulatory networks (GRNs) from single-cell transcriptomic data is crucial for biological discovery, yet existing approaches suffer from a fundamental misalignment with real-world needs. Researchers typically seek a small set of high-confidence regulatory interactions for experimental validation, often involving previously unseen genes. However, current benchmarks rely on transductive splits with global classification metrics, while prevailing models struggle to generalize under inductive settings. To bridge this gap, we reformulate GRN inference as an inductive, ranking-centric graph completion problem and introduce \benchmark, a new benchmark that incorporates an inductive gene-holdout split together with knowledge graph completion metrics to better evaluate top-ranked predictions. Building on this, we propose \method, the first co-evolutionary discrete diffusion framework that jointly models biologically coherent discretized gene expression states and regulatory interactions for robust inductive generalization and improved top-ranked regulatory discovery. We further introduce TF-ALL Subgraph Sampling (TASS) for scalable training. Extensive experiments on {\benchmark} show that {\method} establishes new state-of-the-art performance, significantly outperforming existing methods in novel regulatory discovery, and ablation studies further verify the effectiveness of our design.

Gene perturbation models aim to predict transcriptional responses to unseen genetic interventions, but comparisons rely on heterogeneous evaluation metrics and differentially-expressed (DE) gene subsets. We investigate the consequences of these choices using eight methods across six single-gene perturbation datasets under a unified evaluation protocol. Varying the number of top DE genes reveals substantial instability in Precision-based rankings, with the winning method changing $86.7\%$ of the time, while rankings based on the rest of the metrics remain comparatively stable. Investigating this discrepancy exposes that repeating a single predicted expression profile across cell-level rows, a standard practice for many methods, can inflate significance estimates and produce excessively large predicted significant-DE sets. Specifically we found that $67.1\%$ of tested perturbation--gene pairs pass the significance threshold despite some of them having minuscule effects. We derive an inflation factor showing that replicated predictions can amplify the standardized Mann--Whitney statistic even after correction for tied ranks, without adding independent information. This artifact changes Precision mainly as the number of DE genes increases because for smaller cutoffs, most of the genes are already significant due to the effect-based ordering. Taking this pitfall into account in our benchmark comparison, we find that a random forest using Gene Ontology features achieves the best overall results.