WhatsGNU protein allele frequencies

WhatsGNU computes the frequency of each protein allele across all bacterial genomes. For every protein in a query genome, the WhatsGNU score (GNU score) reports how many of the 2,438,285 AllTheBacteria genomes carry an identical copy of that protein sequence.

This is useful for identifying conserved versus rare alleles, understanding species-level protein diversity, and contextualising novel genomes against all publicly available bacterial data.

What is available

The pre-built WhatsGNU database covers 2,438,285 genomes from AllTheBacteria (release 0.2 plus incremental release 2024-08). It is built from the Bakta protein annotations (.faa files).

The following files are available on the WhatsGNU OSF component:

Folder	Description
`WGNU_ATB_DB/`	Pre-built LMDB database (8 count shards, 8 posting shards, genome-to-species index, function lookup table, Sample-to-ID mapping, build metadata). Required for querying.
`Sample_tables/`	List of included genomes (`final_2438285_genomes.txt`), species statistics, and per-genome/per-species allele record counts.
`ATB_hash_seq/`	Hash-to-amino-acid-sequence lookup table, split into 20 xz-compressed parts (`hash_to_sequence_part_00.xz` - `part_19.xz`).
`ATB_summary_figures_tables/`	Publication figures, per-species GNU histograms, allele frequency tables, species-sharing networks, coverage estimates, cross-species allele analyses, and the pre-computed counts cache.
`panallelome_summary.txt`	Summary results for 2.4M genomes.
`panallelome_summary.html`	Summary results with selected summary figures for 2.4M genomes.

Installation

Option A - Conda (recommended, once available on bioconda)

conda install -c bioconda whatsgnu-atb

Option B - pip

pip install whatsgnu-atb

Option C - From source

git clone https://github.com/microbialARC/WhatsGNU-ATB.git
bash WhatsGNU-ATB/setup_whatsgnu_atb.sh
conda activate whatsgnu-atb

Downloading the database

Use the included downloader to fetch data from OSF. No OSF account or token is required - the project is public.

Download the database (required for querying):

python WhatsGNU-ATB/scripts/download_osf.py \
    --folder WGNU_ATB_DB \
    --out-dir ./WGNU_ATB_DB

Download everything:

python WhatsGNU-ATB/scripts/download_osf.py \
    --all \
    --out-dir ./WGNU_ATB_DB

List available folders:

python WhatsGNU-ATB/scripts/download_osf.py --list

The downloader skips files that have already been downloaded with the correct size, so it is safe to rerun if a download is interrupted.

Querying a genome

Your input must be a protein FASTA (.faa) file. If your genome has not yet been annotated with Bakta, see the Annotation documentation.

Basic query (GNU scores only)

python WhatsGNU-ATB/scripts/Query_WhatsGNU_ATB.py \
    --db_dir WGNU_ATB_DB \
    --shards 8 \
    --faa your_genome.bakta.faa \
    --out_dir results/

This produces your_genome.bakta.faa.whatsgnu.tsv with one row per protein, including its BLAKE2b hash and GNU score.

Full query (with species breakdown and genome similarity)

python WhatsGNU-ATB/scripts/Query_WhatsGNU_ATB.py \
    --db_dir WGNU_ATB_DB \
    --shards 8 \
    --faa your_genome.bakta.faa \
    --include_sequence \
    --with_postings \
    --samples_tsv WGNU_ATB_DB/samples_with_ids.tsv \
    --species_names_tsv WGNU_ATB_DB/samples_with_ids.tsv \
    --top_k_species 5 \
    --top_k_genomes 10 \
    --out_dir results/

Querying multiple genomes

Pass a directory of .faa files instead of a single file:

python WhatsGNU-ATB/scripts/Query_WhatsGNU_ATB.py \
    --db_dir WGNU_ATB_DB \
    --shards 8 \
    --faa directory_of_faa_files/ \
    --include_sequence \
    --with_postings \
    --out_dir results_batch/

Output files

File	Description
`<sample>.whatsgnu.tsv`	Per-protein results: protein ID, BLAKE2b allele hash, GNU score (allele frequency across 2.4M genomes), and optionally the amino acid sequence and top species contributing to each allele.
`<sample>.similarity.tsv`	Genome similarity report: ranks the genomes in AllTheBacteria that share the most protein alleles with the query, with species information and percentage of query proteome shared (requires `--with_postings`).

Interpreting GNU scores

The GNU score for a protein is the number of genomes (out of 2,438,285) that contain an identical copy of that exact amino acid sequence:

High GNU score (e.g. >100,000): a highly conserved allele found across many genomes and likely multiple species.
Moderate GNU score (e.g. 1000-100,000): a common allele, often species- or genus-specific.
Low GNU score (e.g. 1-100): a rare allele, possibly strain-specific or recently evolved.
GNU score = 0: unique to your query genome - not found in any other AllTheBacteria genome.

Hash-to-sequence lookup

The ATB_hash_seq/ folder contains the mapping from each allele hash to its full amino acid sequence, split into 20 compressed parts. To reassemble:

python WhatsGNU-ATB/scripts/download_osf.py \
    --folder ATB_hash_seq \
    --out-dir ./whatsgnu_db

cd whatsgnu_db/ATB_hash_seq/
cat hash_to_sequence_part_*.xz | xz -d > hash_to_sequence.tsv

Building a custom database

To build a new database from your own set of genomes, use WhatsGNU_ATB_DB.py. See the GitHub repository for full build documentation, including resumable builds, local scratch options, and all available parameters.

Reproducibility

Full methods, commands, software versions, and all scripts used to build the AllTheBacteria WhatsGNU database are available in the GitHub reproducibility directory.

Citation

If you use WhatsGNU-ATB in your research, please cite:

Moustafa AM and Planet PJ. WhatsGNU: a tool for identifying proteomic novelty. Genome Biology, 2020. doi:10.1186/s13059-020-01965-w
AllTheBacteria - all bacterial genomes assembled, available and searchable. bioRxiv, 2024. doi:10.1101/2024.03.08.584059

Hypothetical protein structures Species specific typing