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bioRxiv · 10.64898/2025.12.04.692398

Uniform pre-processing of bacterial single-cell RNA-seq

Oakes, C. G.·Beilinson, V.·McFall-Ngai, M. J.·Pachter, L. G.
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Abstract

Bacteria are highly heterogeneous, even under controlled conditions, making single-cell RNA sequencing (scRNA-seq) essential for studying microbial diversity and symbiosis. Since its first application in 2015, bacterial scRNA-seq has expanded, but different assays depend on distinct, custom, in-house preprocessing making it difficult to analyze data as part of a unified workflow. The kallisto-bustools suite of tools has enabled uniform pre-processing of eukaryotic scRNA-seq while also reducing time and resource demands for pre-processing, but is not optimized for bacterial scRNA-seq. We adapt kallisto-bustools to be suitable for reads generated from operons, as well as for a much shorter gene length distribution, and show that it can efficiently and accurately quantify bacterial scRNA-seq. Our work provides a scalable foundation for uniform pre-processing and analysis of microbial single-cell transcriptomics.

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Oakes, C. G., Beilinson, V., McFall-Ngai, M. J., Pachter, L. G.. 2025-12-06. Uniform pre-processing of bacterial single-cell RNA-seq. https://doi.org/10.64898/2025.12.04.692398

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