Version Compatibility

Reference examples tested with: kallisto 0.50+, 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
• 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.

Splicing Quantification

Quantify alternative splicing events as PSI (percent spliced in) values from RNA-seq data.

Event Types

| Type | Code | Description | |------|------|-------------| | Skipped exon | SE | Exon inclusion/exclusion | | Alternative 5' splice site | A5SS | Alternative donor site | | Alternative 3' splice site | A3SS | Alternative acceptor site | | Mutually exclusive exons | MXE | One of two exons included | | Retained intron | RI | Intron retention |

Tool Selection

SUPPA2 (transcript TPM-based)

• Input: Transcript TPM from Salmon/kallisto
• Faster, requires transcript quantification
• Better for isoform-level analysis

rMATS-turbo (BAM-based)

• Input: Aligned BAM files
• Junction read counting
• Better for novel junction discovery

SUPPA2 Workflow

Goal: Calculate PSI values for all splicing event types from transcript-level quantification.

Approach: Generate event definitions from GTF annotation, then compute per-event PSI from transcript TPM using SUPPA2.

"Quantify splicing from RNA-seq" -> Extract splicing events from annotation, then calculate inclusion ratios from transcript abundance.

• Python/CLI: suppa.py generateEvents + suppa.py psiPerEvent (SUPPA2)
• CLI: rmats.py with --statoff (rMATS-turbo, BAM-based)

python
import subprocess
import pandas as pd

gtf_file = 'annotation.gtf'
tpm_file = 'transcript_tpm.tsv'
output_prefix = 'events'

# Step 1: Generate splicing events from annotation
subprocess.run([
    'suppa.py', 'generateEvents',
    '-i', gtf_file,
    '-o', output_prefix,
    '-f', 'ioe',  # IOE format for PSI calculation
    '-e', 'SE', 'SS', 'MX', 'RI', 'FL'  # All event types
], check=True)

# Step 2: Calculate PSI values
for event_type in ['SE', 'A5', 'A3', 'MX', 'RI']:
    ioe_file = f'{output_prefix}_{event_type}_strict.ioe'
    subprocess.run([
        'suppa.py', 'psiPerEvent',
        '-i', ioe_file,
        '-e', tpm_file,
        '-o', f'psi_{event_type}'
    ], check=True)

# Load and examine PSI values
psi_se = pd.read_csv('psi_SE.psi', sep='\t', index_col=0)
print(f'Quantified {len(psi_se)} skipped exon events')
print(psi_se.head())

rMATS-turbo Workflow

Goal: Quantify splicing events directly from aligned BAM files using junction read counting.

Approach: Run rMATS-turbo on paired BAM groups with annotation, then parse inclusion level columns from output.

bash
# rMATS-turbo for BAM-based quantification
rmats.py \
    --b1 condition1_bams.txt \
    --b2 condition2_bams.txt \
    --gtf annotation.gtf \
    -t paired \
    --readLength 150 \
    --nthread 8 \
    --od output_dir \
    --tmp tmp_dir \
    --statoff  # Use for quantification only, no differential testing

python
import pandas as pd

# Load rMATS output
se_jc = pd.read_csv('output_dir/SE.MATS.JC.txt', sep='\t')

# Calculate average PSI across samples
# IncLevel columns contain PSI values per sample
inc_cols = [c for c in se_jc.columns if c.startswith('IncLevel')]
se_jc['mean_PSI'] = se_jc[inc_cols].mean(axis=1)

# Filter for reliable events (sufficient junction reads)
# Minimum 10-20 junction reads recommended for reliable PSI
se_jc['total_junction_reads'] = se_jc['IJC_SAMPLE_1'] + se_jc['SJC_SAMPLE_1']
reliable_events = se_jc[se_jc['total_junction_reads'] >= 20]
print(f'{len(reliable_events)} events with sufficient coverage')

Quality Thresholds

| Metric | Threshold | Rationale | |--------|-----------|-----------| | Junction reads | >= 10-20 | Minimum for reliable PSI estimation | | PSI range | 0.1-0.9 | Events outside this range are nearly constitutive | | Missing values | < 50% samples | High missingness indicates low expression |

Output Interpretation

PSI values range from 0 to 1:

• PSI = 1.0: Event fully included (e.g., exon always present)
• PSI = 0.5: Equal inclusion/exclusion
• PSI = 0.0: Event fully excluded (e.g., exon always skipped)

Related Skills

• differential-splicing - Compare PSI between conditions
• rna-quantification/alignment-free-quant - Generate transcript TPM for SUPPA2
• read-alignment/star-alignment - Align reads with junction detection