BacSC: A general workflow for bacterial single-cell RNA sequencing data analysis

Bacterial single-cell RNA sequencing has the potential to elucidate within-population heterogeneity of prokaryotes, as well as their interaction with host systems. Despite conceptual similarities, the statistical properties of bacterial single-cell datasets are highly dependent on the protocol, making proper processing essential to tap their full potential. We present BacSC, a fully data-driven computational pipeline that processes bacterial single-cell data without requiring manual intervention. BacSC performs data-adaptive quality control and variance stabilization, selects suitable parameters for dimension reduction, neighborhood embedding, and clustering, and provides false discovery rate control in differential gene expression testing. We validated BacSC on a broad selection of bacterial single-cell datasets spanning multiple protocols and species. Here, BacSC detected subpopulations in Klebsiella pneumoniae, found matching structures of Pseudomonas aeruginosa under regular and low-iron conditions, and better represented subpopulation dynamics of Bacillus subtilis. BacSC thus simplifies statistical processing of bacterial single-cell data and reduces the danger of incorrect processing.