ZoonomiX

ZoonomiX is a comprehensive bioinformatics pipeline designed to analyze microbial genomes for identifying critical genes and predicting their roles in antibiotic resistance, virulence, pathogenicity, and zoonotic potential. Leveraging BLASTn and curated reference databases, ZoonomiX evaluates sequences to score future risks and adaptability of microbes.

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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 for their potential pathogenicity and zoonotic risk.

• Comprehensive Analysis:

  • Utilizes curated databases:
    • Adherence
    • Biofilm
    • Efflux Pumps
    • Exotoxins
    • Integrative and Conjugative Elements (ICEs)
    • Secretion Systems (T3SS, T4SS, T6SS)
    • Zoonotic Virulence Factors
  • Incorporates horizontal gene transfer (HGT) markers like ICEs and resistance elements.

• Customizable Parameters:

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

• Detailed Outputs:

  • Tabular report with gene annotations, alignment statistics, and risk scores.
  • Summarized Excel file 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 and 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.
Virulance_genes_Zoonotic	13	Zoonotic virulence factors.

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Prerequisites:

1. Hardware Requirements:

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

2. Software Requirements:

Python Environment

• Python 3.8 or later.

• Required Python Libraries:

  • pandas: For data manipulation and analysis.
  • openpyxl: For Excel file handling.
  • biopython: For biological sequence data processing.

• Install libraries using pip:

pip install pandas openpyxl biopython

BLAST+ (Basic Local Alignment Search Tool)

Download and install BLAST+ from the NCBI BLAST website.

Add BLAST executables (e.g., blastn, makeblastdb) to your system PATH.

Test installation:

Copy code

blastn -version

Spreadsheet Software Microsoft Excel or alternatives like LibreOffice or Google Sheets to view output files. Text Editor/IDE

Recommended editors:

• VSCode
• PyCharm
• Any plain text editor.

Query Sequences File:

A properly formatted FASTA file containing microbial sequences (e.g., query_sequences.fasta).

Reference Database:

A BLAST-compatible database created using makeblastdb from BLAST+.

Optional Annotation File:

Excel file for mapping categories (e.g., annotations_1.xlsx).

Output Files

Annotated Results: .tsv file with BLAST+ results annotated by category.

Excel Summary:

Summarizes gene categories, alignment statistics, risk scores, and pathogenicity levels.

Setup Instructions

1. Set Up BLAST+

Download BLAST+ tools from the NCBI BLAST website.

Unzip the downloaded file and add the BLAST executable directory to your system's PATH.

Test the installation:

blastn -version

2. Prepare Input Files

Ensure the FASTA file for query sequences is formatted correctly.

Create a BLAST-compatible reference database using makeblastdb:

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

The reference_db.nsq file can be accessed from this link.

Here is the direct link to access the reference_db.nsq file: https://drive.google.com/file/d/1Tw67zxHtDQePQsi3ZtrZQoB9sqc9NFk-/view?usp=drive_link.

3. Verify Python Environment

Install Python 3.8 or later.

Install required Python libraries:

pip install pandas openpyxl biopython

4. Run the Pipeline

Modify the script to match local file paths.

Execute the script from the terminal:

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

License:

ZoonomiX is free to use and share.

BLAST+ (Basic Local Alignment Search Tool), a core component of this pipeline, is also free to use and distributed under the terms of the Public Domain Notice from the National Center for Biotechnology Information (NCBI). For more details, visit the NCBI BLAST+ License.

Users are encouraged to comply with any licenses for third-party tools or data used alongside this pipeline.

Contact

For inquiries, suggestions, or collaborations, please contact me at research.umeshkumarku@gmail.com or open an issue in this repository.