sanger_targseq

A comprehensive Nextflow pipeline for automated analysis of Sanger sequencing data, from raw .ab1 trace files to pathogen identification and phylogenetic analysis.

🔬 Overview

This pipeline streamlines the analysis of Sanger sequencing data for veterinary pathogen detection, providing:

• Consensus assembly from .ab1 trace files
• Pathogen typing using custom databases
• BLAST-based identification
• Phylogenetic analysis with multiple sequence alignment and tree construction
• Comprehensive HTML reports with visualizations

✨ Features

• 📊 Tracy Assembly: Assembles .ab1 trace files into high-quality consensus sequences
• 🔍 BLAST Analysis: Performs searches on both consensus sequences and individual reads
• 🧬 Pathogen Typing: Custom abricate database screening for selected veterinary pathogen detection and serotyping
• 🌳 Phylogenetic Analysis: Multiple sequence alignment (MAFFT) and phylogenetic tree construction (IQ-TREE)
• 📈 Automated Reporting: Generates comprehensive HTML reports with results and visualizations

🚀 Quick Start

Prerequisites

• Nextflow (≥22.12.0-edge)
• Docker
• WSL2 (for Windows users)

Basic Usage

# Clone the repository
git clone https://github.com/dhineshp565/sanger_targseq.git
cd sanger_targseq

# Run the pipeline
nextflow run main.nf --input /path/to/input --out_dir /path/to/output

Input Structure

Your input directory should contain subdirectories, each with .ab1 trace files and reads files in txt or seq format:

input_directory/
├── sample1/
│   ├── trace1.ab1
│   ├── trace1.txt          
│   ├── trace2.ab1
│   ├── trace2.seq          
│   └── trace3.ab1
├── sample2/
│   ├── trace1.ab1
│   ├── trace1.txt          
│   ├── trace2.ab1
│   └── trace2.txt          
└── sample3/
    ├── trace1.ab1
    └── trace1.seq         

File Types:

• Required: .ab1 - Sanger sequencing trace files for consensus assembly .txt or .seq - Associated reads files containing sequence data

⚙️ Parameters

Required Parameters

Parameter	Description	Example
--input	Path to input directory containing subdirectories with .ab1 files	/data/sanger_data/
--out_dir	Output directory path	/results/sanger_analysis/
--blastdb_path	Path to BLAST database	/data/referenceDB/blast/microbe_db
--blastdb_name	Name of BLAST database	microbe_db

Example Commands

# Basic run
nextflow run main.nf --input /data/samples --out_dir /results

# With custom BLAST database
nextflow run main.nf \
  --input /data/samples \
  --out_dir /results \
  --blastdb_path /custom/blast/db \
  --blastdb_name custom_db

📁 Output Structure

output_directory/
├── tracy_assembly/          # Consensus sequences and alignment files
├── abricate/               # Pathogen typing results
├── blast_consensus/        # BLAST results for consensus sequences
├── blast_sanger_reads/     # BLAST results for individual reads
├── orfipy/                # ORF predictions
├── seq_length/            # Sequence length statistics
├── mafft/                 # Multiple sequence alignments
├── iqtree/                # Phylogenetic trees
├── ggtree/                # Tree visualizations
├── LIMS/                  # Laboratory information management files
├── execution/             # Pipeline execution reports
└── sanger_targseq_report.html  # Main results report

🛠️ Pipeline Workflow

1. Input Processing: Creates sample list from input directory structure
2. Tracy Assembly: Assembles .ab1 files into consensus sequences
3. Quality Control: Generates reverse complement sequences for analysis
4. Pathogen Typing: Screens sequences against veterinary pathogen database
5. BLAST Analysis: Searches against reference databases
6. ORF Prediction: Identifies open reading frames
7. Phylogenetic Analysis: Multiple alignment and tree construction
8. Reporting: Generates comprehensive HTML reports with visualizations

🔧 Dependencies

Core Tools (Containerized)

• Tracy (v0.7.8): Sanger trace file assembly
• Abricate (v1.0.1): Antimicrobial resistance gene detection
• BLAST+ (v2.16.0): Sequence similarity search
• MAFFT (v7.526): Multiple sequence alignment
• IQ-TREE (v2.4.0): Phylogenetic tree reconstruction
• OrfIpy (v0.0.4): ORF prediction
• R/RMarkdown (v2.10): Report generation
• seqtk (v1.4): Sequence processing utilities

🐛 Troubleshooting

Common Issues

1. Input directory structure: Verify subdirectories contain .ab1 files
2. Permission errors: Check file/directory permissions

�️ Software Used

This pipeline integrates the following software tools:

Core Analysis Tools

• Tracy (v0.7.8) - Sanger trace file basecalling, alignment, and assembly
• Abricate (v1.0.1) - Mass screening of contigs for antimicrobial resistance and virulence genes
• BLAST+ (v2.16.0) - Basic Local Alignment Search Tool for sequence similarity searches
• MAFFT (v7.526) - Multiple sequence alignment program
• IQ-TREE (v2.4.0) - Efficient phylogenomic software for maximum likelihood phylogenetic inference
• seqtk (v1.4) - Toolkit for processing sequences in FASTA/FASTQ formats

Additional Tools

• OrfIpy (v0.0.4) - Fast and flexible tool for extracting ORFs from nucleotide sequences
• R with RMarkdown (v2.10) - Statistical computing and dynamic report generation
• ggtree - Phylogenetic tree visualization and annotation

Workflow Management

• Nextflow (≥22.12.0-edge) - Workflow management system
• Docker - Containerization platform for reproducible environments

Container Images

All tools are containerized using Docker images from:

• Quay.io Biocontainers
• Docker Hub official repositories
• StaPH-B (State Public Health Bioinformatics) collection
• NCBI official images

Note: Exact container versions and sources can be found in the config/docker.config file.

📝 License

This project is open source and available under the MIT License.

👤 Author

dhineshp565

• GitHub: @dhineshp565
• Pipeline: sanger_targseq

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Version: v1.0.6
Last Updated: September 2025