Splicing Quantification

What the source does

• FilesystemNone observedNo reads or writes in the source
• NetworkNone observedNo outbound calls in the source
• ShellNone observedNo commands run in the source
• DependenciesPinnedNothing floats
• SecretsNone foundNo keys in source

Five audit layers

Gates applied: no behavioural pass.

Audit 0.4.1 · source sha c8c1fc3a2b13 · 19 Oct 2026Report an issue or request a re-scan

• PublisherGPTomics GitHub organizationOwner of the repository this skill is read from
• Source folderGPTomics/bioSkills/alternative-splicing/splicing-quantificationThe exact folder we audited
• LicenceMITAs the repository states it
• Commit pinned for this verdictc8c1fc3a2b13A new audit runs when the source changes
• Repository statusArchivedLast push 65 days ago
• Sandboxed runNot runSkills are read, not executed; layer L4 is skipped

Its description: Quantifies alternative splicing as PSI (percent spliced in) from RNA-seq using rMATS-turbo (BAM-based event), SUPPA2 (TPM-based event), MAJIQ V3 (LSV-based Bayesian), leafcutter (annotation-free intron clusters),…

---
name: bio-splicing-quantification
description: Quantifies alternative splicing as PSI (percent spliced in) from RNA-seq using rMATS-turbo (BAM-based event), SUPPA2 (TPM-based event), MAJIQ V3 (LSV-based Bayesian), leafcutter (annotation-free intron clusters), VAST-TOOLS (cross-species with microexon support), Shiba (junction-imbalance-corrected, 2025 SOTA at low coverage), or IRFinder-S (intron retention coverage-aware). Distinguishes the five canonical event classes (SE, A5SS, A3SS, MXE, RI), special classes (microexons, exitrons, AFE/ALE), intron retention subtypes (canonical RI vs detained introns), and applies effective-length normalization. Use when measuring splice-site usage or isoform inclusion ratios from short-read RNA-seq.
tool_type: mixed
primary_tool: rMATS-turbo
---

## Version Compatibility

Reference examples tested with: rMATS-turbo 4.3+, SUPPA2 2.4+, leafcutter 0.2.9+, MAJIQ 3.0+, IRFinder-S 2.0+, kallisto 0.50+, Salmon 1.10+, pandas 2.2+, STAR 2.7.11+

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.

# Splicing Quantification

Quantify alternative splicing events as PSI (percent spliced in) from RNA-seq. PSI = inclusion read evidence / (inclusion + skipping read evidence), normalized for differential mapping opportunity between isoforms. The choice of *quantification unit* (event, intron cluster, LSV, transcript) determines which biological questions can be answered and which failure modes apply.

## Algorithmic Taxonomy

| Family | Unit | Reference tools | Fails when |
|--------|------|-----------------|------------|
| Event-based | Pre-defined SE/A5SS/A3SS/MXE/RI events from annotation | rMATS-turbo, SUPPA2, VAST-TOOLS | Event isn't in annotation; complex multi-junction events split arbitrarily; AFE/ALE confounded with splicing |
| LSV-based | Local Splice Variations at single source/target nodes | MAJIQ V3 | Memory-constrained environments; cohorts smaller than ~3 reps; non-academic users (license) |
| Junction-cluster | Annotation-free intron clusters by shared splice sites | leafcutter, leafcutter2 | Undersampled clusters lose power; topology biologically uninterpretable for novel events |
| Splice-graph | Graph nodes (non-overlapping exonic regions) | Whippet, Shiba | Whippet maintenance status uncertain since ~2022; complex multi-exon graphs |
| Coverage-aware (IR) | Intron body coverage + flanking junctions | IRFinder-S, S-IRFindeR, iREAD | Confounded by overlapping exons, repeats, low mappability regions |
| Isoform-based | Transcript abundance via EM | Salmon/kallisto + tximport | Salmon EM uncertainty propagates; many similar isoforms (TTN, MAPT) become indistinguishable |

The agent's first decision is **which family** the question requires, not which tool. Switching family is the response to a tool failing within a family — switching tool within a family rarely fixes systematic blind spots.

## Event Taxonomy (Beyond Standard SE/A5SS/A3SS/MXE/RI)

| Class | Code | Biology | Detection caveat |
|-------|------|---------|-------------------|
| Skipped exon (cassette) | SE | Default cassette-exon AS | Most common; well-handled by all tools |
| Alternative 5' splice site | A5SS | Alternative donor; intron 5' end varies | Sign convention tool-specific (see below) |
| Alternative 3' splice site | A3SS | Alternative acceptor; intron 3' end varies | Sensitive to BPS cancer mutations (SF3B1) — cryptic 3'ss ~10-30nt upstream |
| Mutually exclusive exons | MXE | Two exons paired, one included | Tool implementations vary; verify which "form 1" is yours |
| Retained intron | RI | Whole intron retained in mature mRNA | Junction-only quant systematically underdetects; needs IRFinder-S |
| **Microexon** | (sub-SE) | 3-27 nt; neural-enriched, SRRM4-regulated | Missed by default aligner anchor lengths (>=20-30 nt); needs VAST-TOOLS, MicroExonator, or long-read |
| **Exitron** | n/a | Intronic region within an annotated CDS exon | Mis-classified as A5SS/A3SS by most tools; use ExitronFinder, ScanExitron |
| **Alternative first exon (AFE)** | AFE | Alternative TSS / promoter use | Promoter-driven, NOT spliceosomal; confirm with FANTOM CAGE before reporting as splicing |
| **Alternative last exon (ALE)** | ALE | Alternative cleavage/polyadenylation | APA-driven, NOT spliceosomal; confirm with 3'-end-seq |
| **Detained intron (DI)** | (RI subtype) | Nuclear-retained on mature mRNA, regulated by Clk kinases | Distinct from cytoplasmic NMD-targeted RI (Boutz 2015 *Genes Dev*); requires fractionation to confirm |
| **Recursive splicing** | (intron subtype) | Long introns >50kb spliced via internal ratchet points | Sibley 2015 *Nature*; only detectable with long-read or nascent RNA-seq |

Tool-agnostic taxonomy reference: Wang 2008 *Nature*; Vaquero-Garcia 2016 *eLife* (LSV); Tapial 2017 *Genome Res* (VastDB).

## Tool Selection Matrix

| Tool | Best for | Input | Strengths | Fails when |
|------|----------|-------|-----------|------------|
| rMATS-turbo | Standard SE/A5SS/A3SS/MXE/RI in annotated organism, n>=3 | BAM + GTF | Fast, well-calibrated at n>=3, novel SS support | Junction read imbalance; novel multi-junction events; underdetects RI and microexons |
| SUPPA2 | Quick PSI from existing TPM, pilot analysis | Salmon/kallisto TPM + GTF | No alignment; fastest | Annotation-bound; high FDR (15-30%) at n<=2 vs n<=2 |
| MAJIQ V3 | Complex events, heterogeneous cohorts (HET) | BAM + GFF3 | Bayesian posterior PSI, complete LSV semantics | High memory (~50+ GB on cohorts); academic license; complex LSV interpretation needs care |
| leafcutter | Novel junction discovery, sQTL, 

Excerpt, first 6,000 characters of SKILL.md, verbatim from the audited commit. It is text from someone else’s repository, quoted so you can judge it before your agent reads it.

pip install suppa pandas

git clone https://github.com/GPTomics/bioSkills
git -C bioSkills checkout c8c1fc3a2b13

Works in

1. 2026-10-19
   ✓SAFE B 89 / 100

   first audit · source c8c1fc3a2b13

Questions about Splicing Quantification

• github.com/GPTomics/bioSkills/tree/main/alternative-splicing/spl — the audited source at commit c8c1fc3a2b13 and every finding aboveObserved · 19 Oct 2026

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