A-T.50: Igniting full-length isoform analysis in single-cell and spatial RNA-seq data with FLAMESv2
Recent advancements in long-read single-cell and spatial RNA-sequencing enable the profiling of RNA isoform expression and alternative splicing at unprecedented resolution. However, the computational landscape remains highly fragmented. Existing pipelines are often tied to specific protocols, require matched short-read data, or lack complete end-to-end processing. These constraints severely limit analytical flexibility, reproducibility, and the ability to compare findings across studies utilizing different methodologies.
To alleviate these computational limitations, we introduce FLAMESv2, a highly modular and protocol-agnostic R/Bioconductor package for the comprehensive analysis of long-read single-cell and spatial RNA-seq data. Building upon our previous framework, FLAMESv2 has been extensively enhanced to offer superior flexibility, processing speed, and usability. It integrates diverse data types and supports a wide array of single-cell workflows as well as emerging spatial transcriptomics protocols. The pipeline is highly configurable, scales efficiently to accommodate multi-sample analyses, and can be executed using long reads alone or in combination with short reads. Furthermore, comprehensive benchmarking confirms that FLAMESv2 achieves field-leading performance across key analysis tasks, from accurate isoform identification to precise quantification. Beyond data processing, FLAMESv2 equips users with versatile built-in functions for publication-ready data visualization and downstream analysis. By transforming a disjointed set of bioinformatic tools into a unified, robust pipeline, FLAMESv2 provides the community with a powerful approach to long-read transcriptomics. It unlocks the full potential of single-cell and spatial long-read sequencing, empowering researchers to deeply characterize the hidden layers of RNA isoform regulation in health and disease.
Co-authors: Yair D.J. Prawer, Matthew E. Ritchie, Michael B. Clark, Yupei You
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