Igniting full-length isoform analysis in single-cell and spatial RNA-seq data with FLAMESv2

Wang, C.; Prawer, Y. D. J.; Voogd, O.; Schuster, J.; De Paoli-Iseppi, R.; Li, A.; Hallab, J.; Tian, L.; Peng, H.; David, M.; Du, M. R. M.; Velasco, S.; Garone, M. G.; Dong, X.; Zeglinski, K.; Pavan, C.; Law, K. C. L.; Abu-Bonsrah, K. D.; Hunt, C. P. J.; Parish, C.; Gouil, Q.; Thijssen, R.; Davidson, N. M.; Ritchie, M. E.; Clark, M. B.; You, Y.

doi:10.1101/2025.10.19.683327

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bioRxiv · 10.1101/2025.10.19.683327

Igniting full-length isoform analysis in single-cell and spatial RNA-seq data with FLAMESv2

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Abstract

Long-read single-cell RNA-sequencing enables the profiling of RNA isoform expression and alternative splicing at single cell resolution. However, diverse single-cell technologies and sparse isoform data demand flexible and accurate analysis tools. We introduce FLAMESv2, a highly modular and protocol-agnostic R/Bioconductor package for long-read single-cell RNA-seq data analysis. FLAMESv2 supports a wide range of single-cell and spatial protocols, is highly configurable, scales to allow multi-sample analysis and provides versatile visualisation and analysis outputs. We demonstrate its compatibility with both droplet-based and combinatorial barcoding single-cell methods, as well as spatial transcriptomics workflows. Benchmarking confirms FLAMESv2 achieves field-leading performance across key analysis tasks. Applying FLAMESv2 to in vitro differentiation of stem cells into neurons, we identify cell-types, differentiation trajectories, expression of annotated and novel isoforms and isoform expression diversity and heterogeneity within individual cells. FLAMESv2 provides a comprehensive, flexible approach to analysing long-read single-cell RNA-sequencing, unlocking this powerful methodology for RNA isoform characterisation.

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Wang, C., Prawer, Y. D. J., Voogd, O., Schuster, J., De Paoli-Iseppi, R., Li, A., Hallab, J., Tian, L., Peng, H., David, M., Du, M. R. M., Velasco, S., Garone, M. G., Dong, X., Zeglinski, K., Pavan, C., Law, K. C. L., Abu-Bonsrah, K. D., Hunt, C. P. J., Parish, C., Gouil, Q., Thijssen, R., Davidson, N. M., Ritchie, M. E., Clark, M. B., You, Y.. 2025-10-20. Igniting full-length isoform analysis in single-cell and spatial RNA-seq data with FLAMESv2. https://doi.org/10.1101/2025.10.19.683327

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