tools-only Splicing Pipeline - Usage Guide
Overview
Complete alternative splicing analysis workflow from raw RNA-seq FASTQ files to differential splicing results and visualizations. Includes QC checkpoints and best practices.
Prerequisites
# Core tools
conda install -c bioconda star rmats-turbo ggsashimi fastp rseqc
# Python dependencies
pip install pandas numpy matplotlib
# R packages (optional, for isoform switching)
BiocManager::install('IsoformSwitchAnalyzeR')
Quick Start
Tell your AI agent what you want to do:
- "Run a complete splicing analysis from my FASTQ files"
- "Set up a splicing pipeline for my RNA-seq experiment"
- "Analyze differential splicing between my conditions"
- "Generate sashimi plots for my significant splicing events"
Example Prompts
Full Pipeline
"I have RNA-seq FASTQs from 3 control and 3 treatment samples. Run a complete splicing analysis."
"Set up STAR 2-pass alignment and rMATS differential splicing for my samples."
Individual Steps
"Align my samples with STAR 2-pass mode for optimal junction detection."
"Run junction saturation QC to check if I have enough sequencing depth."
"Visualize the top 20 differential splicing events with sashimi plots."
What the Agent Will Do
- Perform read QC and adapter trimming (fastp)
- Align with STAR 2-pass mode for junction detection
- Run junction saturation QC checkpoint
- Perform differential splicing analysis (rMATS-turbo)
- Filter significant events (FDR < 0.05, |deltaPSI| > 0.1)
- Optionally analyze isoform switches
- Generate sashimi plots for visualization
Tips
- STAR 2-pass mode is essential for novel junction discovery
- Check junction saturation curves before trusting results
- Use
--outSJfilterOverhangMin 8 8 8 8for stringent junction filtering - rMATS-turbo combines quantification and differential testing
- Standard thresholds: |deltaPSI| > 0.1, FDR < 0.05
- Always require minimum junction reads (>= 10) for reliability
Related Skills
- alternative-splicing/splicing-quantification - Quantification details
- alternative-splicing/differential-splicing - Analysis methods
- alternative-splicing/sashimi-plots - Visualization
- read-alignment/star-alignment - STAR alignment options