Cell clustering is crucial for uncovering cellular heterogeneity in single-cell RNA sequencing (scRNA-seq) data by identifying cell types and marker genes. Despite its importance, existing benchmarks for scRNA-seq clustering remain fragmented, lacking standardized protocols and often omitting recent advances in artificial intelligence.To fill these gaps, we present scCluBench, a comprehensive benchmark of clustering algorithms for scRNA-seq data. scCluBench provides 36 scRNA-seq datasets collected from diverse public sources, covering multiple tissues, which are uniformly processed to ensure consistency for systematic evaluation and downstream analyses. To assess performance, we collect and reproduce a range of scRNA-seq clustering methods, including traditional, deep learning-based, graph-based, and biological foundation models. We comprehensively evaluate each method both quantitatively and qualitatively, using core performance metrics and visualization analyses. Furthermore, we construct representative downstream biological tasks, such as marker gene identification and cell type annotation, to further assess the practical utility. scCluBench then investigates the performance differences and applicability boundaries of various clustering models across diverse analytical tasks, systematically assessing their robustness and scalability in real-world scenarios. Overall, scCluBench offers a standardized and user-friendly benchmark for scRNA-seq clustering, with standardized datasets, unified evaluation protocols, and transparent analyses, facilitating informed method selection and providing valuable insights into model generalizability and application scope.

Single-cell RNA sequencing (scRNA-seq) reveals cell heterogeneity, with cell clustering playing a key role in identifying cell types and marker genes. Recent advances, especially graph neural networks (GNNs)-based methods, have significantly improved clustering performance. However, the analysis of scRNA-seq data remains challenging due to noise, sparsity, and high dimensionality. Compounding these challenges, GNNs often suffer from over-smoothing, limiting their ability to capture complex biological information. In response, we propose scSiameseClu, a novel Siamese Clustering framework for interpreting single-cell RNA-seq data, comprising of 3 key steps: (1) Dual Augmentation Module, which applies biologically informed perturbations to the gene expression matrix and cell graph relationships to enhance representation robustness; (2) Siamese Fusion Module, which combines cross-correlation refinement and adaptive information fusion to capture complex cellular relationships while mitigating over-smoothing; and (3) Optimal Transport Clustering, which utilizes Sinkhorn distance to efficiently align cluster assignments with predefined proportions while maintaining balance. Comprehensive evaluations on seven real-world datasets demonstrate that scSiameseClu outperforms state-of-the-art methods in single-cell clustering, cell type annotation, and cell type classification, providing a powerful tool for scRNA-seq data interpretation.

BACKGROUND: Clustering analysis is fundamental in single-cell RNA sequencing (scRNA-seq) data analysis for elucidating cellular heterogeneity and diversity. Recent graph-based scRNA-seq clustering methods, particularly graph neural networks (GNNs), have significantly improved in tackling the challenges of high-dimension, high-sparsity, and frequent dropout events that lead to ambiguous cell population boundaries. However, one major challenge for GNN-based methods is their reliance on hard graph constructions derived from similarity matrices. These constructions introduce difficulties when applied to scRNA-seq data due to: (i) The simplification of intercellular relationships into binary edges (0 or 1) by applying thresholds, which restricts the capture of continuous similarity features among cells and leads to significant information loss. (ii) The presence of significant inter-cluster connections within hard graphs, which can confuse GNN methods that rely heavily on graph structures, potentially causing erroneous message propagation and biased clustering outcomes. RESULTS: To tackle these challenges, we introduce scSGC, a Soft Graph Clustering for single-cell RNA sequencing data, which aims to more accurately characterize continuous similarities among cells through non-binary edge weights, thereby mitigating the limitations of rigid data structures. The scSGC framework comprises three core components: (i) a zero-inflated negative binomial (ZINB)-based feature autoencoder designed to effectively handle the sparsity and dropout issues in scRNA-seq data; (ii) a dual-channel cut-informed soft graph embedding module, constructed through deep graph-cut information, capturing continuous similarities between cells while preserving the intrinsic data structures of scRNA-seq; and (iii) an optimal transport-based clustering optimization module, achieving optimal delineation of cell populations while maintaining high biological relevance. CONCLUSION: By integrating dual-channel cut-informed soft graph representation learning, a ZINB-based feature autoencoder, and optimal transport-driven clustering optimization, scSGC effectively overcomes the challenges associated with traditional hard graph constructions in GNN methods. Extensive experiments across ten datasets demonstrate that scSGC outperforms 13 state-of-the-art clustering models in clustering accuracy, cell type annotation, and computational efficiency. These results highlight its substantial potential to advance scRNA-seq data analysis and deepen our understanding of cellular heterogeneity.