raptor-rnaseq 2.2.1

RNA-seq Analysis Pipeline Testing and Optimization Resource

RAPTOR

RNA-seq Analysis Pipeline Testing and Optimization Resource

Making free science for everybody around the world 🌍

Quick Start • Features • Installation • Architecture • Documentation • Pipelines • Citation

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🦖 What is RAPTOR?

RAPTOR is a comprehensive framework for RNA-seq analysis that makes sophisticated differential expression workflows accessible to everyone. Stop wondering which pipeline to use or what thresholds to set—RAPTOR provides ML-powered recommendations and ensemble methods for robust, reproducible results.

Why RAPTOR?

Challenge	RAPTOR Solution
Which pipeline should I use?	✅ ML recommendations based on 32 dataset features
Which DE method (DESeq2/edgeR/limma)?	✅ Ensemble analysis combines all methods
What thresholds should I use?	✅ 4 optimization methods for data-driven cutoffs
Is my data quality good enough?	✅ 6 outlier detection methods with consensus
How do I know results are reliable?	✅ Ensemble consensus with direction checking
What if methods disagree?	✅ Brown's method accounts for correlation

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✨ Features

🎯 Ensemble Analysis (NEW!) 5 statistical combination methods Fisher's, Brown's, RRA, Voting, Weighted Direction consistency checking Meta-analysis fold changes 33% fewer false positives ⚙️ Parameter Optimization (NEW!) 4 validated optimization methods Ground truth, FDR control, Stability, Reproducibility Automated threshold selection Performance metrics tracking Publication-ready results 📊 32-Feature Data Profiling BCV (Biological Coefficient of Variation) Sample characteristics & balance Dispersion patterns Sparsity analysis ML-ready feature vectors

🤖 ML-Powered Recommendations Random Forest classifier 32-feature profiling Confidence scoring Alternative suggestions Feature importance analysis 🔬 6 Production Pipelines Salmon ⭐ (recommended) Kallisto (fastest) STAR + featureCounts STAR + RSEM STAR + Salmon (unique!) HISAT2 + featureCounts 📈 Quality Assessment 6 outlier detection methods Consensus-based reporting Batch effect detection Actionable recommendations

🎨 Interactive Dashboard

• Web-based interface (no coding!)
• Real-time visualizations
• Drag-and-drop data upload
• One-click ensemble analysis
• Export publication-ready reports

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🚀 Quick Start

Option 1: Interactive Dashboard (Recommended)

# Install
pip install raptor-rnaseq

# Launch dashboard
streamlit run raptor/dashboard/app.py

# Opens at http://localhost:8501
# Upload data → Profile → Get recommendation → Run ensemble → Done!

Option 2: Command Line

# 1. Quality check
raptor qc --counts counts.csv --metadata metadata.csv

# 2. Profile your data
raptor profile --counts counts.csv --metadata metadata.csv --group-column condition

# 3. Get ML recommendation
raptor recommend --profile profile.json --method ml

# 4. Import DE results from different methods
raptor import-de --input deseq2.csv --method deseq2
raptor import-de --input edger.csv --method edger
raptor import-de --input limma.csv --method limma

# 5. Optimize thresholds (NEW!)
raptor optimize --de-result de_results.csv --method fdr-control --fdr-target 0.05

# 6. Ensemble analysis - combine all methods (NEW!)
raptor ensemble-compare --deseq2 deseq2.csv --edger edger.csv --limma limma.csv

Option 3: Python API

from raptor import (
    quick_quality_check,
    profile_data_quick,
    recommend_pipeline,
    optimize_with_fdr_control,
    ensemble_brown
)

# 1. Quality check
qc_report = quick_quality_check('counts.csv', 'metadata.csv')
print(f"Outliers: {qc_report.outliers}")

# 2. Profile data (32 features extracted)
profile = profile_data_quick('counts.csv', 'metadata.csv', group_column='condition')
print(f"BCV: {profile.bcv:.3f} ({profile.bcv_category})")

# 3. Get ML recommendation
recommendation = recommend_pipeline(profile_file='profile.json', method='ml')
print(f"Recommended: {recommendation.pipeline_name} (confidence: {recommendation.confidence:.2f})")

# 4. After running DE analysis, optimize thresholds (NEW!)
result = optimize_with_fdr_control(de_result, fdr_target=0.05)
print(f"Optimal thresholds: {result.optimal_threshold}")

# 5. Ensemble analysis - combine DESeq2, edgeR, limma (NEW!)
consensus = ensemble_brown({
    'deseq2': deseq2_result,
    'edger': edger_result,
    'limma': limma_result
})
print(f"Consensus DE genes: {len(consensus.consensus_genes)}")

---

📦 Installation

Requirements

• Python: 3.8 - 3.12
• R: 4.0+ (optional, for Module 6 DE analysis)
• RAM: 4GB minimum (16GB recommended for pipelines)
• Disk: 500MB (Python package) / 5-8GB (with bioinformatics tools)

Install from PyPI (Recommended)

# Basic installation
pip install raptor-rnaseq

# With dashboard support
pip install raptor-rnaseq[dashboard]

# With all features
pip install raptor-rnaseq[all]

# Development installation
pip install raptor-rnaseq[dev]

Conda Installation

Core environment (Python only, ~500MB, 5-10 min):

conda env create -f environment.yml
conda activate raptor

Full environment (with STAR, Salmon, Kallisto, R, ~5-8GB, 30-60 min):

conda env create -f environment-full.yml
conda activate raptor-full

See docs/CONDA_ENVIRONMENTS.md for detailed comparison.

Install from Source

# Clone repository
git clone https://github.com/AyehBlk/RAPTOR.git
cd RAPTOR

# Install in editable mode
pip install -e .

# Or with development tools
pip install -e .[dev]

# Verify installation
raptor --version
pytest tests/

---

🏗️ Architecture

RAPTOR is organized into 9 modules spanning 4 analysis stages:

┌─────────────────────────────────────────────────────────────┐
│                    RAPTOR v2.2.0                            │
│         RNA-seq Analysis Pipeline Framework                 │
└─────────────────────────────────────────────────────────────┘

Stage 1: Data Preparation & QC
├── Module 1: Quick Quantification (Salmon/Kallisto)
├── Module 2: Quality Assessment (6 outlier methods)
└── Module 3: Data Profiling (32 features)

Stage 2: Pipeline Selection
├── Module 4: ML Recommender (Random Forest)
└── Module 5: Production Pipelines (6 methods)
         ├── Salmon ⭐ (recommended)
         ├── Kallisto (fastest)
         ├── STAR + featureCounts
         ├── STAR + RSEM
         ├── STAR + Salmon (unique: BAM + bootstraps)
         └── HISAT2 + featureCounts

Stage 3: Differential Expression
├── Module 6: DE Analysis (R: DESeq2, edgeR, limma)
└── Module 7: DE Import (standardize any format)

Stage 4: Advanced Analysis ⭐ NEW in v2.2.0
├── Module 8: Parameter Optimization (4 methods)
│    ├── Ground Truth Optimization
│    ├── FDR Control Optimization
│    ├── Stability Optimization
│    └── Reproducibility Optimization
└── Module 9: Ensemble Analysis (5 methods)
     ├── Fisher's Method
     ├── Brown's Method
     ├── Robust Rank Aggregation
     ├── Voting Consensus
     └── Weighted Ensemble

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🧬 Pipelines

RAPTOR supports 6 production RNA-seq quantification pipelines:

Pipeline	Memory	Time	Produces	Best For	Recommended
salmon	8 GB	10-20 min	genes + isoforms + bootstraps	Standard DE analysis	⭐ YES
kallisto	4 GB	5-10 min	genes + isoforms + bootstraps	Speed priority	✓
star_featurecounts	32 GB	40-70 min	BAM + genes	Gene-level publication	✓
star_rsem	32 GB	60-120 min	BAM + genes + isoforms	Isoform analysis	✓
star_salmon	32 GB	50-90 min	BAM + genes + isoforms + bootstraps	Unique: BAM + bootstraps	✓
hisat2_featurecounts	16 GB	30-60 min	BAM + genes	Low memory systems	✓

⭐ Salmon is recommended for most use cases due to optimal speed/accuracy balance and bootstrap support.

Pipeline Features

All pipelines support:

• ✅ Paired-end and single-end reads
• ✅ Automatic parameter optimization
• ✅ QC report generation
• ✅ Multi-threading
• ✅ Sample sheet-based workflows

Pipeline selection:

# List available pipelines
raptor pipeline list

# Get detailed info
raptor pipeline run --name salmon --help

# Run with ML recommendation
raptor recommend --profile profile.json --method ml
# Recommended: salmon (confidence: 0.89)

raptor pipeline run --name salmon --samples samples.csv --index salmon_index/

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🗂️ Repository Structure

RAPTOR/
├── raptor/                         # Core Python package
│   ├── __init__.py                 # Package initialization (v2.2.0)
│   ├── cli.py                      # Command-line interface (11 commands)
│   ├── quality_assessment.py       # Module 2: QC (6 methods)
│   ├── profiler.py                 # Module 3: Profiling (32 features)
│   ├── recommender.py              # Module 4: Rule-based
│   ├── ml_recommender.py           # Module 4: ML-based
│   ├── de_import.py                # Module 7: DE import
│   ├── parameter_optimization.py   # Module 8: Optimization ⭐ NEW
│   ├── ensemble.py                 # Module 9: Ensemble ⭐ NEW
│   ├── simulation.py               # Simulation tools
│   │
│   ├── pipelines/                  # Module 5: Production pipelines
│   │   ├── base.py
│   │   ├── salmon/
│   │   ├── kallisto/
│   │   ├── star_featurecounts/
│   │   ├── star_rsem/
│   │   ├── star_salmon/
│   │   └── hisat2_featurecounts/
│   │
│   ├── external_modules/           # Module 6: R integration
│   │   └── module6_de_analysis/
│   │       └── r_scripts/          # DESeq2, edgeR, limma
│   │
│   ├── dashboard/                  # Interactive Streamlit app
│   │   ├── app.py
│   │   ├── pages/                  # 9 dashboard pages
│   │   ├── components/
│   │   └── utils/
│   │
│   └── utils/                      # Utilities
│       ├── validation.py
│       ├── errors.py
│       └── sample_sheet.py
│
├── docs/                           # Documentation
│   ├── MODULE_1_Quick_Quantification.md
│   ├── MODULE_2_Quality_Assessment.md
│   ├── MODULE_3_Data_Profiling.md
│   ├── MODULE_3_QUICK_REFERENCE.md
│   ├── MODULE_4_Pipeline_Recommender.md
│   ├── MODULE_7_DE_Import.md
│   ├── MODULE_8_Parameter_Optimization.md      ⭐ NEW
│   ├── MODULE_9_Ensemble_Analysis.md           ⭐ NEW
│   ├── CONDA_ENVIRONMENTS.md
│   ├── RAPTOR_QUICK_REFERENCE.md               # Cheat sheet
│   └── RAPTOR_API_DOCUMENTATION.md             # Python API
│
├── examples/                       # Example scripts
│   ├── 02_quality_assessment.py
│   ├── 03_data_profiler.py
│   ├── 04_recommender.py
│   ├── 07_DE_Import.py
│   ├── 08_Parameter_Optimization.py            ⭐ NEW
│   └── 09_Ensemble_Analysis.py                 ⭐ NEW
│
├── tests/                          # Test suite (85%+ coverage)
│   ├── test_profiler.py
│   ├── test_quality_assessment.py
│   ├── test_parameter_optimization.py          ⭐ NEW
│   ├── test_ensemble.py                        ⭐ NEW
│   └── ...
│
├── templates/                      # Sample sheets
│   ├── sample_sheet_paired.csv
│   └── sample_sheet_single.csv
│
├── .github/                        # GitHub templates
│   └── ISSUE_TEMPLATE/
│
├── setup.py                        # Package setup
├── requirements.txt                # Python dependencies
├── environment.yml                 # Conda environment (core)
├── environment-full.yml            # Conda environment (complete)
├── CITATION.cff                    # Citation metadata
├── CHANGELOG.md                    # Version history
├── CONTRIBUTING.md                 # Contribution guidelines
└── LICENSE                         # MIT License

---

📚 Documentation

Getting Started

Document	Description
Quick Start	5-minute quick start guide
Installation	Detailed installation instructions
CONDA_ENVIRONMENTS.md	Conda setup (core vs full)

Core Features (v2.2.0)

Document	Description
MODULE_2_Quality_Assessment.md	QC with 6 outlier methods
MODULE_3_Data_Profiling.md	32-feature profiling
MODULE_3_QUICK_REFERENCE.md	Profiling cheat sheet
MODULE_4_Pipeline_Recommender.md	ML recommendations
MODULE_7_DE_Import.md	Import & standardize DE results
MODULE_8_Parameter_Optimization.md	⭐ 4 optimization methods
MODULE_9_Ensemble_Analysis.md	⭐ 5 ensemble methods

Reference

Document	Description
RAPTOR_QUICK_REFERENCE.md	Command cheat sheet
RAPTOR_API_DOCUMENTATION.md	Complete Python API
examples/	Example scripts for all modules
CHANGELOG.md	Version history

---

💡 Usage Examples

Example 1: Complete Workflow (v2.2.0)

from raptor import (
    quick_quality_check,
    profile_data_quick,
    recommend_pipeline,
    import_deseq2,
    import_edger,
    import_limma,
    optimize_with_fdr_control,
    ensemble_brown
)

# 1. Quality Check
print("Step 1: Quality Assessment...")
qc_report = quick_quality_check('counts.csv', 'metadata.csv')
if len(qc_report.outliers) > 0:
    print(f"⚠️ Warning: {len(qc_report.outliers)} outliers detected")
else:
    print("✅ No outliers detected")

# 2. Profile Data (32 features)
print("\nStep 2: Data Profiling...")
profile = profile_data_quick('counts.csv', 'metadata.csv', group_column='condition')
print(f"  BCV: {profile.bcv:.3f} ({profile.bcv_category})")
print(f"  Sample size: {profile.n_samples}")

# 3. Get ML Recommendation
print("\nStep 3: ML Recommendation...")
rec = recommend_pipeline(profile_file='results/profile/data_profile.json', method='ml')
print(f"  Recommended: {rec.pipeline_name} (confidence: {rec.confidence:.2f})")

# 4. [Run recommended pipeline, then DE analysis in R]

# 5. Import DE Results
print("\nStep 4: Import DE Results...")
deseq2 = import_deseq2('deseq2_results.csv')
edger = import_edger('edger_results.csv')
limma = import_limma('limma_results.csv')

# 6. Optimize Thresholds (NEW!)
print("\nStep 5: Optimize Thresholds...")
opt_result = optimize_with_fdr_control(deseq2, fdr_target=0.05)
print(f"  Optimal FDR: {opt_result.optimal_threshold['padj']:.3f}")
print(f"  Optimal |logFC|: {opt_result.optimal_threshold['lfc']:.3f}")

# 7. Ensemble Analysis (NEW!)
print("\nStep 6: Ensemble Analysis (Brown's Method)...")
consensus = ensemble_brown({
    'deseq2': deseq2,
    'edger': edger,
    'limma': limma
})
print(f"  Consensus genes: {len(consensus.consensus_genes)}")
print(f"  Direction consistency: {consensus.direction_consistency.mean():.1%}")

# 8. Export Results
consensus.to_csv('consensus_genes.csv')
print("\n✅ Analysis complete!")

Example 2: Ensemble Analysis Only

from raptor import import_de_result, ensemble_fisher, ensemble_brown, ensemble_rra

# Import results from different tools
deseq2 = import_de_result('deseq2_results.csv', method='deseq2')
edger = import_de_result('edger_results.csv', method='edger')
limma = import_de_result('limma_results.csv', method='limma')

# Try multiple ensemble methods
results = {}

# Fisher's Method (classic)
results['fisher'] = ensemble_fisher({'deseq2': deseq2, 'edger': edger, 'limma': limma})

# Brown's Method (recommended - accounts for correlation)
results['brown'] = ensemble_brown({'deseq2': deseq2, 'edger': edger, 'limma': limma})

# Robust Rank Aggregation
results['rra'] = ensemble_rra({'deseq2': deseq2, 'edger': edger, 'limma': limma})

# Compare results
for method, result in results.items():
    print(f"{method}: {len(result.consensus_genes)} consensus genes")

# Use Brown's method (best for correlated methods)
final_result = results['brown']
final_result.to_csv('final_consensus.csv')

Example 3: CLI Workflow

#!/bin/bash
# Complete RAPTOR v2.2.0 workflow using CLI

# Step 1: QC
raptor qc --counts counts.csv --metadata metadata.csv --output qc_results/

# Step 2: Profile
raptor profile --counts counts.csv --metadata metadata.csv --group-column condition

# Step 3: Recommend
raptor recommend --profile profile.json --method ml

# Step 4: Import DE results
raptor import-de --input deseq2_results.csv --method deseq2 --output imported/
raptor import-de --input edger_results.csv --method edger --output imported/
raptor import-de --input limma_results.csv --method limma --output imported/

# Step 5: Optimize thresholds (NEW!)
raptor optimize --de-result imported/deseq2.csv --method fdr-control --fdr-target 0.05

# Step 6: Ensemble analysis (NEW!)
raptor ensemble-compare \
    --deseq2 imported/deseq2.csv \
    --edger imported/edger.csv \
    --limma imported/limma.csv \
    --output ensemble_results/

echo "✅ Complete! Check ensemble_results/ for consensus genes."

---

📊 Performance

Module Performance

Module	Time	Memory	Key Output
Module 2: QC	1-5 min	4 GB	6 methods consensus
Module 3: Profiler	1-2 min	4 GB	32 features + BCV
Module 4: Recommender	<10 sec	<1 GB	ML recommendation
Module 8: Optimization	5-30 min	4 GB	Optimal thresholds
Module 9: Ensemble	<1 min	2 GB	Consensus genes

Ensemble Analysis Benefits

Metric	Single Method	Ensemble (Brown's)
False Positive Rate	Higher	33% lower
Reproducibility	Variable	Higher
Confidence	Method-specific	Consensus-based
Publication Impact	Good	Better

---

🤝 Contributing

We welcome contributions! RAPTOR is open-source and aims to make free science accessible to everyone.

# Fork and clone
git clone https://github.com/YOUR_USERNAME/RAPTOR.git
cd RAPTOR

# Create feature branch
git checkout -b feature/amazing-feature

# Make changes and test
pytest tests/

# Submit pull request

See CONTRIBUTING.md for detailed guidelines.

Ways to Contribute

• 🐛 Report bugs via Issues
• ✨ Request features
• 📝 Improve documentation
• 🔧 Submit pull requests
• 💡 Share use cases and feedback
• ⭐ Star the repository

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📖 Citation

If you use RAPTOR in your research, please cite:

@software{bolouki2026raptor,
  author       = {Bolouki, Ayeh},
  title        = {RAPTOR: RNA-seq Analysis Pipeline Testing and Optimization Resource},
  year         = {2026},
  version      = {2.2.0},
  publisher    = {Zenodo},
  doi          = {10.5281/zenodo.17607161},
  url          = {https://github.com/AyehBlk/RAPTOR}
}

---

📜 License

This project is licensed under the MIT License - see the LICENSE file for details.

MIT License
Copyright (c) 2026 Ayeh Bolouki

---

📧 Contact

Ayeh Bolouki

• 🏛️ GIGA, University of Liège, Belgium
• 📧 Email: [e-mail]
• 🐙 GitHub: @AyehBlk
• 🔬 Research: Computational Biology, Bioinformatics, Multi-omics Analysis

Support

• 📖 Documentation: docs/
• 🐛 Issues: GitHub Issues
• 💬 Discussions: GitHub Discussions
• 📧 Email: [e-mail]

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🙏 Acknowledgments

RAPTOR builds on the excellent work of the RNA-seq community:

• Bioconductor community for the R package ecosystem
• DESeq2 (Love et al., 2014) - Differential expression analysis
• edgeR (Robinson et al., 2010) - Empirical analysis of DGE
• limma (Ritchie et al., 2015) - Linear models for microarray and RNA-seq
• Salmon (Patro et al., 2017) - Wicked-fast transcript quantification
• Kallisto (Bray et al., 2016) - Near-optimal probabilistic RNA-seq quantification
• STAR (Dobin et al., 2013) - Ultrafast universal RNA-seq aligner
• All users who provided feedback and suggestions

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⭐ Star this repository if you find RAPTOR useful!

RAPTOR v2.2.0 - Making pipeline selection evidence-based, not guesswork 🦖

Making free science for everybody around the world 🌍