Splicing Pipeline - Usage Guide

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

1. Perform read QC and adapter trimming (fastp)
2. Align with STAR 2-pass mode for junction detection
3. Run junction saturation QC checkpoint
4. Perform differential splicing analysis (rMATS-turbo)
5. Filter significant events (FDR < 0.05, |deltaPSI| > 0.1)
6. Optionally analyze isoform switches
7. 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 8 for 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