Argo: species-resolved profiling of antibiotic resistant genes in complex metagenomes through long-read overlapping
Create a new conda environment and install:
conda create -n argo -c bioconda -c conda-forge argo
conda activate argoDownload the database from Zenodo:
wget -qN --show-progress https://zenodo.org/records/12576528/files/database.tar.gz
tar -xvf database.tar.gzIndex the files:
## If you encounter memory issue please consider manually lowering cpu_count or simply set cpu_count=1
cpu_count=$(python -c 'import os; print(os.cpu_count())')
diamond makedb --in database/prot.fa --db database/prot --quiet
diamond makedb --in database/sarg.fa --db database/sarg --quiet
ls database/*.*.fa | sort | xargs -P $cpu_count -I {} bash -c '
filename=${1%.fa*};
filename=${filename##*/};
minimap2 -x map-ont -d database/$filename.mmi ${1} 2> /dev/null;
echo "Indexed <database/$filename.fa>.";' - {}
## remove temporary files to save space
rm -rf database/*.faNote
Argo by default does not assign taxonomic labels to plasmid reads since plasmids can have multiple hosts (for example NZ_OW968330.1). --plasmid forces taxonomic classification of these reads by assigning them to their "most likely" lineages (ties resolved based on the estimated genome copies of species within a sample), but interpretation requires caution.
We provide an example file comprising 10,000 quality-controlled (processed with Porechop and nanoq) prokaryotic reads (fungal and other reads removed with minimap2), randomly selected from the R10.3 mock sample of Loman Lab Mock Community Experiments.
wget -qN --show-progress https://zenodo.org/records/12571849/files/example.fa.gz
argo example.fa.gz -d database -o . --plasmidYou should see:
INFO: Estimating genome copies ...
INFO: ... found 27.375 copies of genomes (bacteria: 27.375; archaea: 0).
INFO: Assigning taxonomy ...
INFO: Reassigning taxonomy ...
INFO: ... found 8 unique species (bacteria: 8; archaea: 0).
INFO: Overlapping ...
INFO: ... median sequence divergence: 0.0519 | initial identity cutoff: 0.9 * 77.03.
INFO: Annotating ARGs ...
INFO: ... candidate HSPs: 11094 | ARG-containing reads: 627.
INFO: Overlapping of ARG-containing reads ...
INFO: ... median sequence divergence of ARG-containing reads: 0.0504 | identity cutoff: 77.40 | low-identity HSPs: 3511.
INFO: Assigning taxonomy ...
INFO: Graph clustering ...
INFO: ... read clusters: 168 | low-subject-cover HSPs: 525 | overlapping HSPs: 6200 | remaining HSPs: 858
INFO: Set covering ...
INFO: ... ARG copies per genome: 28.581 | ARG types: 18 | ARG subtypes: 157 | ARG-carrying species: 8.
INFO: Done.
Output file example.sarg.tsv lists ARG abundance estimates (copy per genome, cpg) by species:
lineage type subtype carrier copy genome abundance
...
...Staphylococcus aureus multidrug norA chromosome 4.495 3.875 1.160
...Staphylococcus aureus multidrug norB chromosome 4.380 3.875 1.130
...Staphylococcus aureus multidrug norC chromosome 5.813 3.875 1.500
...Staphylococcus aureus multidrug sdrM chromosome 3.996 3.875 1.031
...Staphylococcus aureus tetracycline tet(38) chromosome 1.749 3.875 0.451
...Staphylococcus aureus tetracycline tet(L) plasmid 14.994 3.875 3.869
...
Output file example.sarg.json contains detailed annotation information for ARG-containing reads:
{
...
"dab6192d-f9e3-4eb6-ae8c-3d40e787e072": {
"remark": "ARG-containing",
"hit": [
"multidrug@MFS|norA|WP_001041274.1"
],
"lineage": "Bacteria;Bacillota;Bacilli;Staphylococcales;Staphylococcaceae;Staphylococcus;Staphylococcus aureus",
"plasmid": false
},
...
"a9eae701-3767-4a1f-ac00-8945fcde986a": {
"remark": "ARG-containing",
"hit": [
"aminoglycoside|ant(4')-I|WP_001795128.1",
"tetracycline|tet(L)|WP_012779616.1"
],
"lineage": "Bacteria;Bacillota;Bacilli;Staphylococcales;Staphylococcaceae;Staphylococcus;Staphylococcus aureus",
"plasmid": true
},
...
}