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Single-cell ATAC-seq (scATAC) yields sparse data that make conventional analysis challenging. We developed chromVAR (), an R package for analyzing sparse chromatin-accessibility data by estimating gain or loss of accessibility within peaks sharing the same motif or annotation while controlling for technical biases. chromVAR enables accurate clustering of scATAC-seq profiles and characterization of known and de novo sequence motifs associated with variation in chromatin accessibility.