PHREDATOR

Phred-based Quality Assessment Tool for Sequencing Data

A professional, rule-based QC toolkit for NGS (Next-Generation Sequencing) data analysis. Phredator provides intelligent quality control analysis with context-aware recommendations for FastQC reports.

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🌟 Features

• 🔍 Intelligent QC Analysis: Context-aware quality assessment using organism and experiment-specific profiles
• ⚡ Batch Processing: Process hundreds of samples with parallel execution
• 🧬 25+ Organism Profiles: Pre-configured thresholds for human, mouse, E. coli, yeast, and more
• 🧪 Experiment-Specific Rules: Different expectations for WGS, WES, RNA-seq, ChIP-seq, and metagenomics
• 📊 Aggregate Statistics: Mean ± SD for GC%, quality, duplication with outlier detection
• 🎯 Smart Recommendations: Context-aware advice (e.g., "DON'T remove duplicates for RNA-seq")
• 🔤 Fuzzy Matching: Natural input like "Human", "RNA seq", "chip-seq" automatically matched
• 🎨 Professional Output: Subread-style terminal display with color-coded status indicators
• 📦 MultiQC Support: Parse and analyze MultiQC aggregate reports
• 🧪 Automated Fixes: Generate trimming/filtering commands with smart parameter detection

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📋 Table of Contents

• Installation
• Quick Start
• Usage
  • Single File Analysis
  • Batch Processing
  • MultiQC Integration
• Command Reference
• Parameters
• Output Format
• Organism Profiles
• Experiment Types
• Examples
• Development
• Citation
• License

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🚀 Installation

From PyPI (Recommended)

pip install phredator

From Source

git clone https://github.com/tahagill/Phredator.git
cd phredator
pip install -e .

Requirements

• Python 3.8+
• NumPy
• PyYAML

---

⚡ Quick Start

# Single file analysis
phredator parse sample_fastqc.zip --output parsed.json
phredator analyze parsed.json --organism human --experiment-type rnaseq --output analysis.json

# Batch processing (multiple samples)
phredator batch *_fastqc.zip --organism human --experiment-type rnaseq --parallel 4

# List available organisms
phredator list-organisms

# Show usage examples
phredator examples

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📖 Usage

Single File Analysis

Step 1: Parse FastQC output

phredator parse sample1_fastqc.zip --output sample1.json

Step 2: Analyze with organism/experiment profiles

phredator analyze sample1.json \
    --organism human \
    --experiment-type rnaseq \
    --output analysis1.json

Step 3: Generate fix suggestions (optional)

phredator fix analysis1.json \
    --input-reads sample1.fastq.gz \
    --output fixes1.json

Example Output:

  ██████╗ ██╗  ██╗██████╗ ███████╗██████╗  ██████╗ ████████╗ ██████╗ ██████╗ 
  ██╔══██╗██║  ██║██╔══██╗██╔════╝██╔══██╗██╔═══██╗╚══██╔══╝██╔═══██╗██╔══██╗
  ██████╔╝███████║██████╔╝█████╗  ██║  ██║███████║   ██║   ██║   ██║██████╔╝
  ██╔═══╝ ██╔══██║██╔══██╗██╔══╝  ██║  ██║██╔══██║   ██║   ██║   ██║██╔══██╗
  ██║     ██║  ██║██║  ██║███████╗██████╔╝██║  ██║   ██║   ╚██████╔╝██║  ██║
  ╚═╝     ╚═╝  ╚═╝╚═╝  ╚═╝╚══════╝╚═════╝ ╚═╝  ╚═╝   ╚═╝    ╚═════╝ ╚═╝  ╚═╝

//================================\\
||       QC Analysis Report       ||
\\================================//

         Sample : SRR29487748
         Status : WARN
        Profile : Organism: Human (Homo sapiens) | Experiment: RNA Sequencing

//================================\\
||    Quality Assessments         ||
\\================================//

   [PASS] Per Base Quality               : Excellent quality: mean Q=39.4, median Q=40.3
   [PASS] Gc Content                     : Normal GC content: 49.7% (expected ~52.0%)
   [WARN] Duplication Levels             : High duplication: 86.1% (acceptable for RNA-seq)
   [PASS] Adapter Content                : Minimal adapter content: 0.55%
   [PASS] Overrepresented Sequences      : No overrepresented sequences detected

//================================\\
||       Recommendations          ||
\\================================//

   1. High duplication is normal for RNA-seq (abundant transcripts)
   2. DO NOT remove duplicates for RNA-seq - they are real biological signal

Batch Processing

Process multiple samples in one command with aggregate statistics:

Method 1: Direct file list

phredator batch sample1.zip sample2.zip sample3.zip \
    --organism human \
    --experiment-type rnaseq \
    --output-dir batch_results/ \
    --parallel 4

Method 2: Using wildcards

phredator batch results/qc/*_fastqc.zip \
    --organism human \
    --experiment-type chipseq \
    --output-dir chipseq_qc/ \
    --parallel 8

Method 3: From a list file

# Create sample list
ls /path/to/*_fastqc.zip > samples.txt

# Process all
phredator batch samples.txt \
    --organism mouse \
    --experiment-type wgs \
    --parallel 4

Batch Output:

//================================\\
||   Batch Processing Summary     ||
\\================================//

   Processed 8/8 samples:
   [PASS] 5 (62%)
   [WARN] 3 (38%)
   [FAIL] 0 (0%)

//================================\\
||      Sample Details            ||
\\================================//

   [PASS] Sample1_fastqc
      [PASS] Per Base Quality   : Excellent quality: mean Q=39.4
      [PASS] Gc Content          : Normal GC content: 49.7%
      [PASS] Duplication Levels  : Duplication: 81.8% (normal for RNA-seq)
      ...

//================================\\
||      Average Metrics           ||
\\================================//

   GC content   : 49.6% ± 2.6%
   Quality      : Q39.3 ± 0.1
   Duplication  : 81.3% ± 11.1% (normal for RNA-seq)

//================================\\
||      Recommendations           ||
\\================================//

   1. DO NOT remove duplicates for RNA-seq
   2. High duplication is normal for RNA-seq (abundant transcripts)
   3. Monitor for potential contamination

MultiQC Integration

Parse MultiQC aggregate reports:

phredator parse multiqc_data/multiqc_data.json --output multiqc_parsed.json

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📚 Command Reference

phredator parse

Parse FastQC output or MultiQC JSON.

phredator parse <input> [OPTIONS]

Arguments:

• input: Path to FastQC zip/folder or MultiQC JSON file

Options:

• --output FILE: Output JSON file (default: parsed.json)
• --verbose: Enable verbose logging

phredator analyze

Analyze parsed QC data with organism/experiment profiles.

phredator analyze <input_json> [OPTIONS]

Arguments:

• input_json: Path to parsed JSON from parse command

Options:

• --organism NAME: Organism profile (see list-organisms)
• --experiment-type TYPE: Experiment type (wgs, wes, rnaseq, chipseq, metagenomics)
• --expected-gc FLOAT: Override expected GC% (default: from profile)
• --output FILE: Output JSON file (default: analysis.json)
• --verbose: Enable verbose logging

phredator batch

Process multiple samples with parallel execution.

phredator batch <samples...> [OPTIONS]

Arguments:

• samples: FastQC files/zips OR path to sample list file

Options:

• --organism NAME: Organism profile for all samples
• --experiment-type TYPE: Experiment type for all samples
• --output-dir DIR: Output directory (default: batch_output)
• --parallel N: Number of parallel processes (default: 1)
• --verbose: Enable verbose logging

phredator list-organisms

List available organism profiles.

phredator list-organisms [OPTIONS]

Options:

• --detailed: Show full profile details (GC%, assembly, etc.)

phredator examples

Show comprehensive usage examples.

phredator examples

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🔧 Parameters

Organism Profiles

25+ pre-configured organism profiles with expected GC content and quality thresholds:

Organism	Code	GC%	Notes
Human	human	41%	Homo sapiens
Mouse	mouse	42%	Mus musculus
Rat	rat	42%	Rattus norvegicus
Zebrafish	zebrafish	36%	Danio rerio
Drosophila	drosophila	42%	D. melanogaster
C. elegans	celegans	36%	Worm model
Yeast	yeast	38%	S. cerevisiae
E. coli	ecoli	51%	Bacteria
Arabidopsis	arabidopsis	36%	Plant model
COVID-19	covid19	38%	SARS-CoV-2

View all organisms:

phredator list-organisms --detailed

Experiment Types

Different QC expectations for each experiment type:

Type	Code	Key Characteristics
Whole Genome Sequencing	wgs	Low duplication, uniform coverage
Whole Exome Sequencing	wes	Moderate duplication, targeted regions
RNA Sequencing	rnaseq	High duplication OK (abundant transcripts)
ChIP Sequencing	chipseq	Variable duplication, enriched regions
Metagenomics	metagenomics	Variable GC, mixed organisms

Fuzzy matching supported:

• "RNA seq" → rnaseq
• "chip-seq" → chipseq
• "WGS" → wgs
• "Human" → human

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📊 Output Format

JSON Output

All commands produce structured JSON output:

{
  "sample_name": "SRR29487748",
  "overall_status": "WARN",
  "profile_info": "Organism: Human | Experiment: RNA-seq",
  "metrics": {
    "per_base_quality": {
      "status": "PASS",
      "summary": "Excellent quality: mean Q=39.4, median Q=40.3",
      "details": {...}
    },
    "gc_content": {
      "status": "PASS",
      "summary": "Normal GC content: 49.7% (expected ~52.0%)",
      "details": {...}
    }
  },
  "all_recommendations": [
    "High duplication is normal for RNA-seq",
    "DO NOT remove duplicates for RNA-seq"
  ]
}

Log Files

Automatic .log files created alongside JSON outputs with timestamp and full analysis details.

---

💡 Examples

Example 1: RNA-seq Quality Control

# Parse 8 RNA-seq samples
phredator batch ~/rnaseq/results/*_fastqc.zip \
    --organism human \
    --experiment-type rnaseq \
    --output-dir rnaseq_qc \
    --parallel 4

# Output shows:
# - 5 PASS, 3 WARN, 0 FAIL
# - Average GC: 49.6% ± 2.6%
# - Average Quality: Q39.3 ± 0.1
# - Duplication: 81.3% ± 11.1% (normal for RNA-seq)
# - Recommendations: DON'T remove duplicates

Example 2: ChIP-seq with Adapter Detection

phredator batch ~/chipseq/qc/*_fastqc.zip \
    --organism human \
    --experiment-type chipseq \
    --output-dir chipseq_qc \
    --parallel 8

# Output includes:
# - Adapter content warnings
# - Recommendations: "Trim adapters using Cutadapt"
# - Fix commands generated automatically

Example 3: COVID-19 Variant Surveillance

phredator batch sarscov2_samples/*_fastqc.zip \
    --organism covid19 \
    --experiment-type metagenomics \
    --parallel 16

# High quality thresholds (Q30+) enforced
# GC% ~38% expected for SARS-CoV-2

Example 4: MultiQC Integration

# Parse MultiQC aggregate report
phredator parse multiqc_data/multiqc_data.json --output multiqc_summary.json

# Extract metrics for all 100+ samples at once

---

🧪 Development

Running Tests

# Run comprehensive integration test
pytest tests/test_integration.py -v

# Run with sample data
pytest tests/ -v

Project Structure

phredator/
├── phredator/
│   ├── __init__.py
│   ├── __main__.py
│   ├── cli/
│   │   ├── cli.py              # Command-line interface
│   │   └── commands.py
│   ├── parser/
│   │   ├── fastqc_parser.py    # Parse FastQC output
│   │   ├── multiqc_parser.py   # Parse MultiQC JSON
│   │   └── batch_processor.py  # Batch processing engine
│   ├── analyzer/
│   │   └── qc_analyzer.py      # QC analysis engine
│   ├── rules/
│   │   └── qc_rules.py         # Rule-based evaluation
│   ├── fixer/
│   │   └── qc_fixer.py         # Fix suggestion generator
│   ├── config/
│   │   ├── organisms/          # 25+ organism profiles
│   │   └── experiment_types/   # Experiment-specific rules
│   └── utils/
│       └── helpers.py
├── tests/
│   ├── test_integration.py     # Comprehensive integration test
│   └── test_data/              # Sample FastQC data
├── README.md
├── setup.py
├── requirements.txt
└── LICENSE

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📖 Citation

If you use Phredator in your research, please cite:

@software{phredator2025,
  title={Phredator: Intelligent Quality Control for NGS Data},
  author={Ahmad, Taha},
  year={2025},
  publisher={Zenodo},
  doi={10.5281/zenodo.17641057},
  url={https://github.com/tahagill/Phredator}
}

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📝 License

MIT License - see LICENSE file for details.

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🤝 Contributing

Contributions welcome! Please feel free to submit a Pull Request.

1. Fork the repository
2. Create your feature branch (git checkout -b feature/AmazingFeature)
3. Commit your changes (git commit -m 'Add some AmazingFeature')
4. Push to the branch (git push origin feature/AmazingFeature)
5. Open a Pull Request

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🐛 Issues

Found a bug? Have a feature request? Please open an issue on GitHub.

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📧 Contact

• Author: Taha Ahmad
• Email: tahagill99@gmail.com
• GitHub: @tahagill

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🙏 Acknowledgments

• FastQC team for the excellent QC tool
• MultiQC for aggregate reporting
• Subread for terminal output inspiration