Shotgun-GutFish-PRJNA1254096

Shotgun metagenomic profiling of fish gut microbiota using Kraken2, Bracken, and R.

📘 Project Overview

This project presents a complete workflow for shotgun metagenomic analysis of fish gut microbiota based on a sample (SRR33249106) from BioProject PRJNA1254096.
The sample corresponds to Schizothorax biddulphi, a native fish species from the Kizil River (Xinjiang), and was analyzed using:

• Host decontamination with Kneaddata
• Taxonomic classification with Kraken2 and Bracken
• Functional profiling using eggNOG-mapper
• Visualization with R, Krona, and custom barplots

🧪 Workflow Summary

1. Data Preparation

• Downloaded raw reads using prefetch and fastq-dump
• Performed quality control using FastQC
• Removed host reads using Kneaddata with dual references (fish + human)

2. Taxonomic Profiling

• MetaPhlAn 4 was attempted but yielded no usable matches.
• Used Kraken2 and Bracken for robust classification.
• Visualized taxonomic data using Krona and barplots.

3. Functional Profiling

• Protein-coding genes annotated using eggNOG-mapper
• Top KEGG orthologs and pathways extracted and visualized in R

📁 Repository Structure

Shotgun-GutFish-PRJNA1254096/ ├── data/ # Bracken output, annotations, top species list ├── results/ # Final visualizations (PDFs, HTML) ├── scripts/ # Analysis scripts (R, Python, Shell) ├── LICENSE ├── CITATION.cff └── README.md

📊 Results Preview

• 📄 Top10.pdf: Top 10 abundant species (barplot)
• 📄 Top10-KEGG-Pathways.pdf: Top KEGG pathways
• 📄 Top10-Orthologs.pdf: Top KEGG orthologs
• 📄 10FunctionalTerms.pdf: Functional annotation summary
• 🌐 FishSample06_dualclean_krona_named.html: Interactive Krona plot

🔧 Tools & Versions

| Step | Tool | Version | |------|------|---------| | Quality Control | FastQC | 0.11.9 | | Host decontamination | Kneaddata | 0.10.0 | | Taxonomy | Kraken2 + Bracken | Kraken2 v2.1.2, Bracken v2.6.2 | | Annotation | eggNOG-mapper | 2.1.9 | | Visualization | R | 4.3.1 | | Plotting | ggplot2, pheatmap, dplyr, tidyr | latest CRAN |

▶️ How to Reproduce

See scripts/00_workflow_summary.sh for the overview of all steps.
Each R/Python script is self-contained and assumes input from the /data folder.
Output will be stored in /results.

📜 Citation & Reference

If you use this workflow or any component, please cite:

Sarirchi, Somayeh. (2025). Shotgun metagenomic profiling of fish gut microbiota (PRJNA1254096). Zenodo. https://doi.org/10.5281/zenodo.16447049

Also cite the original dataset:

Shotgun metagenomics analysis of gut microbiota of three indigenous fish species from the Kizil River, Xinjiang (PRJNA1254096)

🙋 Contact

Project by Somayeh Sarirchi
ORCID: 0000-0002-2964-8447

📌 Notes

• This is a public reference project for freelance bioinformatics clients and educational purposes.
• Functional network and ARG analysis are possible in follow-up phases.

🔗 DOI