bio-splicing-pipeline

Version Compatibility

Reference examples tested with: STAR 2.7.11+, fastp 0.23+, numpy 1.26+, pandas 2.2+

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• R: packageVersion('<pkg>') then ?function_name to verify parameters
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Alternative Splicing Analysis Pipeline

"Analyze alternative splicing from my RNA-seq data" → Orchestrate STAR alignment, PSI quantification (rMATS-turbo/SUPPA2), differential splicing detection, isoform switching analysis (IsoformSwitchAnalyzeR), sashimi plot visualization, and junction QC.

Complete workflow from raw RNA-seq to differential splicing results.

Pipeline Overview

FASTQ → Read QC → STAR 2-pass → Junction QC → rMATS-turbo → Results → Visualization
                                    ↓
                            (Optional) IsoformSwitchAnalyzeR

Step 1: Read Quality Control

# fastp for adapter trimming and quality filtering
fastp \
    -i sample_R1.fastq.gz \
    -I sample_R2.fastq.gz \
    -o sample_clean_R1.fastq.gz \
    -O sample_clean_R2.fastq.gz \
    --detect_adapter_for_pe \
    --thread 8 \
    -h sample_fastp.html

Step 2: STAR 2-Pass Alignment

# First pass to detect novel junctions
STAR \
    --runThreadN 8 \
    --genomeDir star_index/ \
    --readFilesIn sample_R1.fastq.gz sample_R2.fastq.gz \
    --readFilesCommand zcat \
    --outFileNamePrefix sample_pass1_ \
    --outSAMtype BAM Unsorted \
    --outSJfilterOverhangMin 8 8 8 8 \
    --alignSJDBoverhangMin 1

# Generate new index with discovered junctions
# (Combine SJ.out.tab files from all samples)
cat *_SJ.out.tab > combined_SJ.out.tab

# Second pass with combined junctions
STAR \
    --runThreadN 8 \
    --genomeDir star_index/ \
    --readFilesIn sample_R1.fastq.gz sample_R2.fastq.gz \
    --readFilesCommand zcat \
    --sjdbFileChrStartEnd combined_SJ.out.tab \
    --outFileNamePrefix sample_ \
    --outSAMtype BAM SortedByCoordinate \
    --outSJfilterOverhangMin 8 8 8 8 \
    --alignSJDBoverhangMin 1 \
    --quantMode GeneCounts