Scalable localization of clustering instability for spatial transcriptomics

Standard spatial transcriptomics workflows rely on clustering methods to define regions of interest for downstream analyses, such as differential expression and pathway enrichment. Although researchers are presented with a single partition, most clustering methods are stochastic, meaning their output depends on random algorithmic choices, typically controlled by a seed; thus, repeating the analysis changing only the seed can yield a different result. Spatial transcriptomics data may be especially sensitive to algorithmic perturbations due to high dimensionality, sparsity, and mixed expression profiles at tissue boundaries and transitional zones; however, the extent of seed-dependent variation and its downstream impact have yet to be systematically evaluated. To bridge this gap, we present InSTability, a framework that localizes disagreement among partitions at the observation level. InSTability is built on (1) an interpretable, per-spot score that quantifies the variability of a spot's cluster assignment across partitions, and (2) an algorithm with time complexity linear in the number of spots for a specified number of runs for computing the instability score at all spots. Together, these advances enable spatially-resolved clustering instability maps for large, high-resolution datasets. Applying InSTability to repeated runs of seven established methods (BayesSpace, GraphST, Leiden, Louvain, SEDR, STAGATE, and SpiceMix) on 18 benchmark samples reveals that clustering instability varies across tissue sections, with shared spatial patterns emerging among different methods applied to the same sample. Furthermore, clustering instability correlates with poor agreement against reference annotations and propagates downstream into differential expression analyses, resulting in inconsistent gene sets across seeds. Thus, InSTability can strengthen spatial transcriptomics studies by highlighting regions where clustering is seed-sensitive and further validation is warranted. InSTability is open-source and available at https://github.com/molloy-lab/InSTability.