ONTmompS is a tool to perform in silico Sequence Based Typing (SBT) of Legionella pneumophila long-read/hybrid assemblies.
python ONTmompS.py -a assembly.fasta
Clone this repository and install the dependencies. We recommend installing in a conda (mamba) environment:
git clone https://github.com/marithetland/ONTmompS.git
mamba create -n ontmomps_env -c bioconda -c conda-forge pandas blast emboss biopython
This tool was built as an in silico approach to identify the Sequence Type (ST) of Legionella pneumophila genomes from long-read or hybrid assemblies. It first identifies the mompS1 and mompS2 alleles and then assigns allele numbers and ST. We recommend using long-read or hybrid assemblies that have circular chromosomes when running this tool. It is not intended for short-read assemblies.
Background
Sequence-based typing (SBT) of Legionella pneumophila is a valuable tool in epidemiological studies and outbreak investigations of Legionnaires’ disease. In the L. pneumophila SBT scheme, mompS2 is one of seven genes that determine the ST. The Legionella genome typically contains two copies of mompS (designated mompS1 and mompS2). When they are non-identical, it can be challenging to determine the mompS2 allele, and subsequently the ST, from Illumina sequences, due to the short read-length. Using long-read sequencing from Oxford Nanopore Technologies (ONT) Kit12/Kit9 chemistry and R10.4/R9.4.1 flow cells, together with Trycycler v0.5.3 and Medaka v1.7.2 for long-read assembly and polishing, we were able to identify the mompS2 allele and subsequently the L. pneumophila ST of 81/81 genomes when using this tool.
Citation
If you use ONTmompS, please cite the paper: Krøvel AV, Hetland MAK, Bernhoff E, et al. Long-read sequencing for reliably calling the mompS allele in Legionella pneumophila sequence-based typing. Front. Cell. Infect. Microbiol (2023)
With default settings, two output files are created: LpST_ONTmompS.tsv reports the ST and allele numbers of the 7 SBT loci. mompS_alleles_ONTmompS.tsv reports the mompS alleles. The ST and alleles will be annotated if there are mismatching or missing alleles:
- A complete ST is reported if allele matches are found to all seven SBT genes in the database (e.g. ST560).
- If there are < 3 inexact matches, the nearest matching ST with the number of locus variants (LVs) is reported, e.g. ST560-1LV.
- For allele matches with <100% sequence identity, the nearest matching allele is noted with "*"
- For incomplete coverage of an allele, the nearest matching allele is noted with "?"
- For loci with no allele matches, sequence identity <90% or sequence coverage <80%, the allele number is reported as "-"
It is possible to store the allele sequences to files, using the flags: store_novel_alleles to store only novel allele sequences, store_mompS_alleles to store the mompS allele sequences, or store_all_alleles to store all allele sequences. The files will by default be placed in a folder named ONTmompS_allele_sequences.
usage: ONTmomps.py [-h] [-v] -a ASSEMBLIES [ASSEMBLIES ...] [--db DB]
[--store_mompS_alleles] [--store_novel_alleles]
[--store_all_alleles] [--verbose] [-l LOG]
[--ST_outfile ST_OUTFILE] [--mompS_outfile MOMPS_OUTFILE]
[-o OUTDIR]
In silico SBT of Legionella pneumophila from long-read or hybrid assemblies
optional arguments:
-h, --help show this help message and exit
-v, --version show program's version number and exit
Input options (required):
-a ASSEMBLIES [ASSEMBLIES ...], --assemblies ASSEMBLIES [ASSEMBLIES ...]
FASTA file(s) for assemblies (*.fasta)
Optional flags:
--db DB Provide a path to database location if different than
that provided by this tool.
--store_mompS_alleles
Print mompS alleles to files named
{assembly}_{allele}.fna.
--store_novel_alleles
Print novel alleles to files named
{assembly}_{allele}.fna.
--store_all_alleles Print all alleles (7 genes in SBT scheme + mompS1) to
files named {assembly}_{allele}.fna.
--verbose Log more details and keep intermediate files for
debugging.
-l LOG, --log LOG Write logging to specified file name instead of stdout
Output options:
--ST_outfile ST_OUTFILE
Output filename for STs. Default: ./LpST_ONTmompS.tsv
--mompS_outfile MOMPS_OUTFILE
Output filename for mompS copy allele numbers.
Default: ./mompS_alleles_ONTmompS.tsv
-o OUTDIR, --outdir OUTDIR
Output directory to store novel alleles in. Default is
current working directory
The database in this repository is the same version as that in https://github.com/tseemann/legsta/tree/master/db. Please contact the Legionella-SBT team at UKHSA if you want to obtain a more recent database version. When you have your desired database, you need to make sure the files follow the same format as those in the db for this repo. If the sequences are provided in csv format with 'sequence,number', you can run the commands below to make the database compatible with ONTmompS:
unzip sbt_schema_10_11_2022_12_59.zip ; cd sbt_schema_10_11_2022_12_59
cat sbt.csv | sed 's/st/ST/g' | sed 's/,/\t/g' >> lpneumophila.txt ; rm sbt.csv
cat neuAh.csv >> neuA.csv ; rm neuAh.csv
for f in $(ls *.csv | sed 's/.csv//g') ; do paste <(cat ${f}.csv | cut -d"," -f2 | sed "s/^/>${f}_/g" ) <(cat ${f}.csv | cut -d"," -f1) | sed 's/\t/\n/g' | grep -v "number\|sequence" >> ${f}.fna ; done ; rm *.csv
Once you have converted the database files, you can either 1) specify the path to your new db with the flag --db /path/to/db or 2) move your new db files to this repo's db directory (i.e. mv *.fna lpneumophila.txt /path/to/ONTmompS/db/).