Reducing batch effects in single cell chromatin accessibility measurements by pooled transposition with MULTI-ATAC

Large-scale scATAC-seq experiments are challenging because of their costs, lengthy protocols, and confounding batch effects. Several sample multiplexing technologies aim to address these challenges, but do not remove batch effects introduced when performing transposition reactions in parallel. We demonstrate that sample-to-sample variability in nuclei-to-Tn5 ratios is a major cause of batch effects and develop MULTI-ATAC, a multiplexing method that pools samples prior to transposition, as a solution. MULTI-ATAC provides high accuracy in sample classification and doublet detection while eliminating batch effects associated with variable nucleus-to-Tn5 ratio. We illustrate the power of MULTI-ATAC by performing a 96-plex multiomic drug assay targeting epigenetic remodelers in a model of primary immune cell activation, uncovering tens of thousands of drug-responsive chromatin regions, cell-type specific effects, and potent differences between matched inhibitors and degraders. MULTI-ATAC therefore enables batch-free and scalable scATAC-seq workflows, providing deeper insights into complex biological processes and potential therapeutic targets.