ZoonomiX 🦠

A Bioinformatics Pipeline for Microbial Genomic Analysis and Risk Prediction

🔬 Introduction

ZoonomiX is a comprehensive bioinformatics pipeline designed to analyze microbial genomes, identify pathogenicity and resistance genes, and detect mobile genetic elements to assess zoonotic risk. By integrating BLASTn for precise scoring and leveraging curated reference databases, ZoonomiX delivers actionable insights for zoonotic risk assessment and advanced genomic research.

This pipeline enables researchers to:

✅ Detect antibiotic resistance genes
✅ Identify virulence factors and pathogenicity markers
✅ Analyze horizontal gene transfer (HGT), including Integrative and Conjugative Elements (ICEs)
✅ Score microbes for zoonotic potential and adaptability

ZoonomiX provides a user-friendly, customizable, and efficient framework for studying microbial evolution, host-pathogen interactions, and genomic epidemiology.

By leveraging BLASTn and curated reference databases, ZoonomiX systematically evaluates microbial adaptability and future risks.

Understanding the genetic makeup of microbes is essential in combating:

• Antimicrobial resistance (AMR)
• Zoonotic diseases
• Emerging infectious threats

ZoonomiX provides a user-friendly, customizable, and efficient solution for researchers studying:

• Microbial evolution
• Host-pathogen interactions
• Horizontal gene transfer (HGT)

🔑 Key Features

✅ Gene Identification

• Detects genes linked to adherence, biofilm formation, efflux pumps, virulence factors, and more.
• Supports custom and curated reference databases for enhanced accuracy.

✅ Resistance and Pathogenicity Prediction

• Identifies genes contributing to antibiotic resistance and virulence.
• Scores microbes based on their pathogenicity and zoonotic risk.

✅ Comprehensive Analysis with Curated Databases

ZoonomiX integrates genetic markers such as:

• Adherence factors
• Biofilm-related genes
• Efflux pumps
• Exotoxins
• Integrative and Conjugative Elements (ICEs)
• Secretion Systems (T3SS, T4SS, T6SS)
• Zoonotic virulence factors

✅ Customizable Parameters

• User-defined thresholds for BLASTn alignment:
  • E-value
  • Percent identity
  • Query coverage

✅ Detailed Outputs

• Tabular reports with gene annotations, alignment statistics, and risk scores.
• Summarized Excel files for easy interpretation.

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📂 Reference Databases

ZoonomiX relies on curated FASTA reference databases:

Database Name	Number of Sequences	Description
Adherence_genes	5,201	Genes related to microbial adherence.
Biofilm_genes	814	Biofilm-related genes aiding persistence.
Efflux_genes	93	Efflux pump genes for multidrug resistance.
Exotoxin_genes	1,324	Genes encoding exotoxins.
ICE_genes	1,326	Genes linked to Integrative & Conjugative Elements.
Resistance_genes	3,151	Known antibiotic resistance genes.
T3SS_genes	925	Genes for Type 3 Secretion Systems.
T4SS_genes	10,795	Genes for Type 4 Secretion Systems.
T6SS_genes	1,411	Genes for Type 6 Secretion Systems.
Virulence_genes_Zoonotic	13	Zoonotic virulence factors.

⚙️ Prerequisites

1️⃣ Hardware Requirements

• A computer with sufficient RAM and storage for bioinformatics tasks.
• Stable internet connection to download tools and databases.

2️⃣ Software Requirements

Python Environment

• Python 3.8 or later
• Install required Python libraries:

  pip install pandas openpyxl biopython
  

🧬 BLAST+ (Basic Local Alignment Search Tool)

1. Download and Install BLAST+

   • Get BLAST+ from the NCBI BLAST website.
   • Extract the downloaded files and install them on your system.

2. Add BLAST executables to system PATH

   • Ensure the BLAST+ executables (blastn, makeblastdb) are accessible from the command line.
   • On Linux/macOS, add the BLAST directory to your .bashrc or .zshrc file:

     export PATH=$PATH:/path/to/blast/bin

   • On Windows, add the BLAST+ installation path to System Environment Variables.

3. Test Installation

   • Open a terminal and check the version:

     blastn -version

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📊 Spreadsheet Software

• Microsoft Excel, LibreOffice, or Google Sheets to view output files.

📝 Text Editor/IDE

• Recommended editors:
  • VSCode
  • PyCharm
  • Any plain text editor

🔧 Setup Instructions

1️⃣ Set Up BLAST+

1. Download BLAST+ from the NCBI BLAST website.
2. Unzip the downloaded file and place it in an accessible directory.
3. Add BLAST+ executables (e.g., blastn, makeblastdb) to your system's PATH.
4. Test the installation by running:

   blastn -version
   

🔧 Setup Instructions

1️⃣ Set Up BLAST+

1. Download BLAST+ from the NCBI BLAST website.
2. Unzip the downloaded file and place it in an accessible directory.
3. Add BLAST+ executables (e.g., blastn, makeblastdb) to your system's PATH.
4. Test the installation by running:

   blastn -version
   

2️⃣ Prepare Input Files

• Ensure the FASTA file for query sequences is formatted correctly.
• Create a BLAST-compatible reference database:

  makeblastdb -in <input_fasta> -dbtype nucl -out reference_db
  

3️⃣ Verify Python Environment

• Install Python 3.8+ and required dependencies:

  pip install pandas openpyxl biopython
  

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▶️ Running ZoonomiX

Modify the script to match your local file paths, then execute:

python zoonomix.py --input query_sequences.fasta --dbpath /path/to/databases --output results.csv