Inference and visualization of multi-scale cell tree for decoding functional diversity with scMustree

Deciphering cellular heterogeneity and functional diversity is crucial to understanding biological systems and disease mechanisms. Although single-cell RNA sequencing (scRNA-seq) has revolutionized this field, existing methods often fail to characterize the functional associations between cell populations due to the discrete nature of clustering results. We introduce scMustree, a tree- construction algorithm that enables in-depth exploration of functional associations and transitional states among cellular populations. scMustree combines top-down iterative decomposition for high- purity leaf nodes with bottom-up merging based on quantitative functional distances, preserving local continuity and global structural clarity. Benchmarking on real datasets shows that scMustree not only outperforms existing methods in clustering accuracy but also reveals functionally coherent subtypes, anatomically related cell types, and developmentally connected transitions. In case studies, it identifies Alzheimer’s disease-associated microglial subtypes and captures spatial cell distribution patterns of cell types in developed embryos, demonstrating its utility as a powerful tool for multi-scale exploration of cellular heterogeneity.