SnakeAltPromoter Facilitates Differential Alternative Promoter Analysis

<b>Background:</b> Alternative promoter usage contributes to isoform diversity and gene regulation in mammals but remains difficult to study at scale. Cap Analysis of Gene Expression precisely maps transcription start sites, but its cost limits large-scale application. Alternatively, ProActiv, Salmon, and DEXSeq can be utilized with widely available RNA sequencing (RNA-seq) data to infer promoter activity. However, there is currently no framework available to automate the generation of reproducible results for these methods. <b>Results:</b> SnakeAltPromoter, a scalable end-to-end Snakemake workflow, has been developed to automate alternative promoter analysis from raw RNA-seq data. The workflow performs quality control, alignment, and promoter quantification using 3 complementary RNA-seq analysis methods (junction-based, transcript-based, and first-exon-based), followed by promoter classification and differential activity or usage analysis. SnakeAltPromoter supports both command-line and graphical user interface usage and utilizes standardized modules to enhance reproducibility. A built-in benchmarking module compares promoter activities inferred from RNA-seq data to matched Cap Analysis of Gene Expression data to evaluate quantification performance. Our analyses revealed robust and complementary performance profiles among the evaluated methods across tissues and cell types, highlighting the value of a unified framework for promoter analysis. <b>Conclusions:</b> To our knowledge, SnakeAltPromoter is the first unified and reproducible framework that combines scalable execution and guided method selection for RNA-seq-based promoter analysis. By standardizing and integrating existing RNA-seq-based promoter analysis tools, it provides researchers with a robust and accessible platform to investigate promoter-level regulation and alternative promoter activity and will enhance the research value of large, public RNA-seq repositories. Code is freely available at https://github.com/YidanSunResearchLab/SnakeAltPromoter.git.