Evolutionary Genomics of Lactobacillales

This repository contains a pipeline to study the evolution of the order Lactobacillales using public genome data.

How to run?

Step 1: clone this repository:

git clone https://github.com/swittouck/legen.git

Step 2: create folders for data and results:

cd legen
mkdir data results

Step 3: download type strain names of validly published species from the LPSN:

1. Go to https://lpsn.dsmz.de/downloads
2. Log in (create an account if you do not yet have one)
3. Download the genera, species and subspecies (GSS) list (e.g. "lpsn_gss_2025-10-03.csv")
4. Put the list in the data folder

Step 4: install all dependencies:

conda env create -f ./environment.yml -p ./env
conda activate ./env

Step 5: install the EggNOG database (required for functional annotation):

download_eggnog_data.py --data_dir data/eggnog

Step 6: run all scripts in src in the order indicated by the file/folder names. Run each script directly from its parent directory. E.g.:

cd src/01_prepare_genomes
./01_download_metadata.R

Step 7: deactivate the conda environment:

conda deactivate

Dependencies

See environment.yml.

LEGEN versions

LEGEN version	GTDB version	year
v3	/	2018
v4	r207	2022
v5	r226	2025

The data

lactobacillales_gtdb-r226.tsv

• metadata of all Lactobacillales genomes that are in release 226 of the GTDB
• downloaded by the script src/01_prepare_genomes/01_download_metadata.R

genomes_selected.txt

• assembly accessions of high-quality genomes
• created by the script src/01_prepare_genomes/02_select_genomes.R

lactobacillales_gtdb-r226

• assemblies (.fna files) of selected genomes
• downloaded by the script src/01_prepare_genomes/03_download_genomes.sh

genomes_failed.txt

• assembly accessions of selected genomes whose download failed
• created by the script src/01_prepare_genomes/03_download_genomes.sh

lpsn_gss_2025-10-03.csv

• type strain names and other info for all validly published species, from LPSN
• downloaded from https://lpsn.dsmz.de/downloads (PNU account required)

eggnog

• the EggNOG database, version 5

Data analyses based on this pipeline

Demonstration of SCARAP and analysis of orthogroup fixation frequency

This analysis was based on v4 of LEGEN and is available in the pangenome-toolkit repository. The results have been published in Bioinformatics:

Wittouck, S., Eilers, T., Van Noort, V., & Lebeer, S. (2024). SCARAP: Scalable cross-species comparative genomics of prokaryotes. Bioinformatics, 41(1), btae735. https://doi.org/10.1093/bioinformatics/btae735

Genus-level taxonomy of Lactobacillaceae

This analysis was based on v3 of LEGEN and is available in the lacto_genus repository. The results have been published in IJSEM:

Zheng, J., Wittouck, S., Salvetti, E., Franz, C. M. A. P., Harris, H. M. B., Mattarelli, P., O’Toole, P. W., Pot, B., Vandamme, P., Walter, J., Watanabe, K., Wuyts, S., Felis, G. E., Gänzle, M. G., & Lebeer, S. (2020). A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. International Journal of Systematic and Evolutionary Microbiology, 70(4), 2782–2858. https://doi.org/10.1099/ijsem.0.004107

Species-level taxonomy of Lactobacillaceae

This analysis was based on v3 of LEGEN and is available in the lacto_species repository. The results have been published in mSystems:

Wittouck, S., Wuyts, S., Meehan, C. J., Van Noort, V., & Lebeer, S. (2019). A genome-based species taxonomy of the lactobacillus genus complex. mSystems, 4(5), e00264-19. https://doi.org/10.1128/mSystems.00264-19