EGGNOG Annotator

Requirements

• Diamond 2.1.16

The annotator uses Diamond to search for matching proteins in the database. The file was created with Diamond v2.1.16. Currently, the software is not bundled with it, so Diamond needs to be installed already on the system. There are no other requirements. Also, the tool has not been tested with other versions of Diamond.

• Linux / Mac

The script makes mainly use of basic bash commands like awk and sed and consequently they need to be isntalled (as it is default on Linux and Mac)

Installation

Tool installation

Just download the eggnog7_annotator.sh script, either by cloning the entire repository or by direct download

My suggestion is to clone it via git and then add the path to your PATH variable to have the commands system-wide available like this

# Go to home folder
cd

# Change to the git repos
mkdir -p git
cd git

# clone it to the folder
git clone https://github.com/fischuu/eggnog7_annotator.git

# Export it to make the command system-wide available
export PATH="/users/fischerd/git/eggnog7_annotator:$PATH"

(Obviously, you need to adjust the first path /users/fischerd/ to the folder to where you cloned repository)

Fetching the databases

Manual download

The eggnog annotator uses a master search table, which was created based on the database files provided by the eggnog project (https://eggnogdb.com) and they can be downloaded directly from our server via wget

# The master search table
wget https://a3s.fi/eggnog7_annotator/eggnog7_20251223_master_search_table.tsv.gz
wget https://a3s.fi/eggnog7_annotator/eggnog7_20251223_master_search_table.tsv.gz.md5
md5sum -c eggnog7_20251223_master_search_table.tsv.gz.md5

# The protein search diamond database
wget https://a3s.fi/eggnog7_annotator/eggnog7_20251223_proteins.dmnd
wget https://a3s.fi/eggnog7_annotator/eggnog7_20251223_proteins.dmnd.md5
md5sum -c eggnog7_20251223_proteins.dmnd.md5

# Optional: test data
wget https://a3s.fi/eggnog7_annotator/test.fa
wget https://a3s.fi/eggnog7_annotator/test.fa.md5
md5sum -c test.fa.md5

Download via wrapper

cd /path/where/the/databases/should/be/fetched
eggnog7_fetchdb.sh

Alternative download server

Apparently, there was some trouble downloading the data from the primary server. A secondary download mirror for the database files is located here

https://doi.org/10.5281/zenodo.18514296

Usage

The script performs a two-step annotation workflow:

1. Run diamond blastp of a protein FASTA against the EggNOG 7 database.
2. Merge Diamond hits with a EggNOG master table to produce a combined annotation file.

All outputs are organized using a sample prefix and an output directory.

---

Basic usage

./eggnog7_annotator.sh [OPTIONS]

Required Arguments

Option	Description
-d FILE	Path to the Diamond database file (.dmnd) downloaded by the user.
-q FILE	Protein FASTA file (amino acid fasta, e.g. from Prodigal).
-m FILE	EggNOG master search table file (.tsv.gz).
-s NAME	Sample name / prefix used for output files (e.g., test).

Optional Arguments

Option	Description	Default
-o DIR	Output directory	. (current directory)
-e FLOAT	E-value threshold for Diamond	1e-5
-p INT	Number of threads	1
--keep-diamond	Keep the intermediate Diamond TSV file	(not kept by default)
-h	Show this help message and exit	-

---

Output Files

All output files are written to <outdir> and use the sample prefix <sample>:

File	Description
<outdir>/<sample>.diamond.tsv	Raw Diamond BLASTP output (optional, deleted by default).
<outdir>/<sample>.eggnog.tsv.gz	Combined annotation file with EggNOG hits.

---

Example

./eggnog7_annotator.sh \
  -d eggnog7_20251223_proteins.dmnd \
  -q test.fa \
  -m eggnog7_20251223_master_search_table.tsv.gz \
  -s test \
  -o results \
  -p 16

After running, the final combined annotation is available at:

results/test.eggnog.tsv.gz

---

Notes

• Make sure the Diamond database and EggNOG master table are downloaded beforehand.
• The script automatically creates the output directory if it does not exist.
• By default, the intermediate Diamond TSV file is removed to save space; use --keep-diamond to preserve it.
• I am not affiliated to the eggNOG project, I just wrote this convenience wrapper for my own usage and share it

If you are searching a worry-free MetaG snakemake pipeline, please have a look at

https://github.com/fischuu/Pipeline-Holoruminant-Meta

References

The Eggnog7 database, upon which these scripts and datatables are based on, is published here

Ana Hernández-Plaza, Ziqi Deng, Fabian Robledo-Yagüe, Damian Szklarczyk, Christian von Mering, Peer Bork, Jaime Huerta-Cepas, eggNOG v7: phylogeny-based orthology predictions and functional annotations, Nucleic Acids Research, Volume 54, Issue D1, 6 January 2026, Pages D402–D408, https://doi.org/10.1093/nar/gkaf1249