autoclustal 1.0.6
Comprehensive bioinformatics pipeline for sequence analysis, clustering, phylogeny, and BLAST annotation
Requires Python: >=3.8
Keywords: bioinformatics, phylogeny, clustering, blast, sequence-analysis, alignment

# AutoClustal: Comprehensive Sequence Analysis Pipeline

AutoClustal is a powerful bioinformatics tool that performs comprehensive sequence analysis including multiple sequence alignment, phylogenetic analysis, clustering, and functional annotation.

## Features

- **Sequence Input**: Support for FASTA and FASTQ formats
- **Sequence Sampling**: Random sampling from large datasets (-M and -N parameters)
- **Automatic Detection**: DNA, RNA, or protein sequence type detection
- **Multiple Alignment**: Integration with MUSCLE, ClustalW, and MAFFT
- **Phylogenetic Analysis**: Distance-based (NJ, UPGMA) and ML methods
- **Sequence Clustering**: Multiple clustering algorithms with representative selection
- **Database Searches**: BLAST and BLAT integration for functional annotation
- **Statistical Analysis**: PCA, t-SNE, and comprehensive reporting (WIP)
- **Advanced Visualizations**: 
  - Phylogenetic tree plots
  - Organism distribution bar charts
  - Model organism analysis (Human, Mouse, C. elegans, Yeast, E. coli, etc.)
  - Cluster analysis plots
  - Publication-ready figures (PNG + PDF)
- **Interactive Reports**: Comprehensive HTML reports with organism statistics

## Installation

### Option 1: Python Virtual Environment (Recommended)

1. **Clone or download the AutoClustal project**
2. **Set up the virtual environment**:
   ```bash
   # Linux/Mac
   ./setup_env.sh
   
   # Windows
   setup_env.bat
   ```

3. **Activate the virtual environment**:
   ```bash
   # Linux/Mac
   source .venv/bin/activate
   
   # Windows
   .venv\Scripts\activate.bat
   ```

### Option 2: Conda Environment

```bash
# Create conda environment
conda env create -f environment.yml

# Activate environment
conda activate autoclustal
```

### Option 3: Manual Installation

#### Requirements

- Python 3.7+
- Required packages (install with `pip install -r requirements.txt`):
  - biopython>=1.81
  - matplotlib>=3.7.0
  - seaborn>=0.12.0
  - pandas>=2.0.0
  - numpy>=1.24.0
  - scikit-learn>=1.3.0
  - scipy>=1.10.0
  - plotly>=5.15.0

### Nextflow Installation (Optional)

For running the Nextflow pipeline, install Nextflow:

```bash
# Install Nextflow
curl -s https://get.nextflow.io | bash
chmod +x nextflow
sudo mv nextflow /usr/local/bin/

# Or on Windows, download from https://www.nextflow.io/
```

### Optional External Tools

For optimal performance, install these external tools:

- **MUSCLE**: Multiple sequence alignment
  - Download from: http://www.drive5.com/muscle/
  - Add to PATH

- **ClustalW**: Alternative alignment tool
  - Download from: http://www.clustal.org/clustal2/
  - Add to PATH

- **MAFFT**: Fast alignment tool
  - Download from: https://mafft.cbrc.jp/alignment/software/
  - Add to PATH

- **BLAST+**: Database searches
  - Download from: https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastDocs&DOC_TYPE=Download
  - Add to PATH

## Quick Start

### Option 1: Python Pipeline

```bash
# Activate virtual environment
source .venv/bin/activate  # Linux/Mac
# or
.venv\Scripts\activate.bat  # Windows

# Run with example sequences
python example.py

# Basic analysis
python autoclustal.py -i sequences.fasta -o results/

# Advanced analysis with BLAST and PCA
python autoclustal.py -i sequences.fasta -o results/ --blast --pca
```

### Option 2: Nextflow Pipeline (WIP)

```bash
# Basic Nextflow run
nextflow run autoclustal.nf --input sequences.fasta --outdir results/

# Advanced analysis
nextflow run autoclustal.nf \
  --input "*.fasta" \
  --outdir results \
  --blast \
  --pca \
  --aligner mafft \
  --phylogeny nj

# Run with specific profile
nextflow run autoclustal.nf --input sequences.fasta -profile docker
```

### Option 3: Visualization Tools

```bash
# Quick visualization of results
python visualize.py results/

# Advanced visualization with custom options
python grapher.py results/ 

# Create demo data for testing
python create_demo_data.py
python visualize.py demo_results/
```

```bash
# Basic Nextflow run
nextflow run autoclustal.nf --input sequences.fasta --outdir results/

# Advanced analysis
nextflow run autoclustal.nf \
  --input "*.fasta" \
  --outdir results \
  --blast \
  --pca \
  --aligner mafft \
  --phylogeny nj

# Run with specific profile
nextflow run autoclustal.nf --input sequences.fasta -profile docker
```

### Command Line Options

#### Python Pipeline
```bash
python autoclustal.py [options]

Required:
  -i, --input           Input FASTA/FASTQ files (supports wildcards)

Optional:
  -o, --output          Output directory (default: autoclustal_results)
  -M, --max-sequences   Maximum number of sequences to take from beginning (default: all)
  -N, --random-sample   Number of sequences to randomly sample from first M (default: all)
  --random-seed         Random seed for reproducible sampling (default: None)
  -t, --type            Sequence type: auto, dna, rna, protein (default: auto)
  -a, --aligner         Alignment tool: muscle, clustalw, mafft (default: muscle)
  -p, --phylogeny       Phylogenetic method: nj, upgma, ml (default: nj)
  --blast               Perform BLAST searches
  --blat                Perform BLAT searches (requires local setup)
  --pca                 Perform PCA analysis
  --cluster-threshold   Clustering threshold (default: 0.8)
  --threads             Number of threads (default: 4)
  --log-level           Logging level: DEBUG, INFO, WARNING, ERROR
```

#### Nextflow Pipeline
```bash
nextflow run autoclustal.nf [options]

Required:
  --input               Input FASTA/FASTQ files

Optional:
  --outdir              Output directory (default: results)
  --seq_type            Sequence type: auto, dna, rna, protein (default: auto)
  --aligner             Alignment tool: muscle, clustalw, mafft (default: muscle)
  --phylogeny           Phylogenetic method: nj, upgma, ml (default: nj)
  --blast               Enable BLAST searches (default: false)
  --blat                Enable BLAT searches (default: false)
  --pca                 Enable PCA analysis (default: false)
  --cluster_threshold   Clustering threshold (default: 0.8)
  --threads             Number of threads (default: 4)
  --max_memory          Maximum memory (default: 16.GB)
  --max_cpus            Maximum CPUs (default: 8)
  --max_time            Maximum time (default: 8.h)
```

### Examples

#### Example 1: Basic DNA sequence analysis
```bash
python autoclustal.py -i dna_sequences.fasta -t dna -a muscle -p nj -o dna_analysis/
```

#### Example 2: Protein analysis with BLAST annotation
```bash
python autoclustal.py -i proteins.fasta -t protein --blast --pca -o protein_analysis/
```

#### Example 3: Multiple files with clustering
```bash
python autoclustal.py -i *.fasta -a mafft --cluster-threshold 0.7 -o multi_analysis/
```

#### Example 4: Large dataset sampling (analyze 100 sequences from first 100,000)
```bash
python autoclustal.py -i huge_dataset.fasta -M 100000 -N 100 --random-seed 42 -o sampled_analysis/
```

#### Example 5: Random sampling from entire dataset
```bash
python autoclustal.py -i large_file.fastq -N 500 --blast --pca -o random_sample_analysis/
```

## Output Files

AutoClustal generates comprehensive output including:

### Data Tables
- `summary_table.csv/html` - Complete analysis summary
- `cluster_statistics.csv` - Cluster-level statistics
- `pca_results.csv` - PCA analysis results (if requested)

### Sequence Analysis
- `alignment.fasta` - Multiple sequence alignment
- `tree.newick` - Phylogenetic tree in Newick format

### Visualizations
- `organism_analysis.png/pdf` - Comprehensive organism distribution plots:
  - Model organism bar chart (Human, Mouse, C. elegans, Yeast, etc.)
  - Organism distribution pie chart
  - Top individual organisms
  - Analysis summary table
- `phylogenetic_tree.png` - Phylogenetic tree visualization
- `cluster_analysis_detailed.png` - Cluster size distributions
- `pca_analysis.png/html` - PCA plots (static and interactive)
- `tsne_analysis.png` - t-SNE visualization
- `annotation_analysis.png` - BLAST/BLAT results summary

### Reports
- `AutoClustal_Report.html` - Comprehensive HTML report
- `analysis_summary.txt` - Text-based summary with organism statistics

## Methodology

### 1. Sequence Processing
- Automatic format detection (FASTA/FASTQ)
- Sequence type detection (DNA/RNA/protein)
- Quality validation and filtering

### 2. Multiple Sequence Alignment
- Support for MUSCLE, ClustalW, and MAFFT
- Automatic parameter optimization
- Alignment quality assessment

### 3. Phylogenetic Analysis
- Distance-based methods (Neighbor-Joining, UPGMA)
- Maximum likelihood (basic implementation)
- Bootstrap support (when available)

### 4. Sequence Clustering
- Distance-based hierarchical clustering
- K-means clustering with optimal k selection
- DBSCAN for density-based clustering
- Representative sequence selection

### 5. Functional Annotation
- BLAST searches against public databases
- Local BLAT searches (when configured)
- Organism identification and classification
- Top hit analysis and statistics

### 6. Statistical Analysis
- Principal Component Analysis (PCA)
- t-SNE for non-linear dimensionality reduction
- Sequence composition analysis
- Clustering validation metrics

## Troubleshooting

### Common Issues

1. **External tools not found**
   - Ensure tools are installed and in PATH
   - Use `which muscle` (Linux/Mac) or `where muscle` (Windows) to verify

2. **BioPython import errors**
   - Install with: `pip install biopython`
   - Update to latest version: `pip install --upgrade biopython`

3. **Memory issues with large datasets**
   - Reduce number of sequences
   - Use sampling for initial analysis
   - Increase system memory

4. **BLAST searches failing**
   - Check internet connection
   - Try local BLAST setup
   - Reduce number of sequences for online searches

### Performance Tips

- Use MAFFT for large alignments (fastest)
- Enable multithreading with `--threads`
- Use representative sequences for initial analysis
- Consider sequence sampling for very large datasets

## API Usage

AutoClustal can also be used as a Python library:

```python
from modules.sequence_handler import SequenceHandler
from modules.alignment import AlignmentEngine
from modules.phylogeny import PhylogeneticAnalyzer
from modules.clustering import SequenceClusterer
from modules.blast_search import BlastSearcher
from modules.analysis import AnalysisReporter

# Initialize components
seq_handler = SequenceHandler()
aligner = AlignmentEngine()
phylo = PhylogeneticAnalyzer()

# Load and process sequences
sequences = seq_handler.load_sequences(['input.fasta'])
alignment = aligner.align_sequences(sequences)
tree = phylo.build_tree(alignment)
```


## License
MIT License

Copyright (C) 2025 Stella R Hartono

Permission is hereby granted, free of charge, to any person obtaining a copy
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The above copyright notice and this permission notice shall be included in all
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