resfinder 4.2.0

ResFinder identifies acquired genes and/or finds chromosomal mutations mediating antimicrobial resistance in total or partial DNA sequence of bacteria.

ResFinder documentation

ResFinder identifies acquired antimicrobial resistance genes in total or partial sequenced isolates of bacteria.

Content of the repository

• run_resfinder.py - Use this script to run ResFinder.
• README.md - This file.
• amr_abbreviations.md - List of antibiotic abbreviations used by ResFinder.
• tests/data - Contains fasta and fastq data for testing.
• scripts/ - All scripts in this directory is unsupported but has been uploaded as they may be useful.
• cge/ - ResFinder code
• dockerfile - Used to build ResFinder docker image (See Docker section near the end)
• database_tests.md - Doctests for use whenever parts of the database has been altered.

Installation

The installation described here will first install the actual ResFinder software, then the dependencies, and finally the databases. A more detailed breakdown of the installation is provided below:

1. Install ResFinder tool
2. Install python modules
3. Install BLAST (optional)
4. install KMA (optional)
5. Download ResFinder database
6. Download PointFinder database
7. Index databases with KMA (if installed)
8. Test installation

A small script has been written to automate this process. It is available from the scripts directory and is named install_resfinder.sh. It is very simple and might not work in all environments. It is only meant as a supplement and no support will be provided for any scripts in this directory. However, specific suggestions (with code) for improvement is very welcome.

ResFinder tool

Setting up ResFinder script and database

# Go to wanted location for resfinder
cd /path/to/some/dir

# Clone the latest version and enter the resfinder directory
git clone https://[e-mail]/genomicepidemiology/resfinder.git
cd resfinder

Dependencies:

Depending on how you plan to run ResFinder BLAST and KMA can be optional. BLAST is used to analyse assemblies (ie. FASTA files). KMA is used to analyse read data (ie. FASTQ files).

Python modules: Tabulate, BioPython, CGECore and CGELib

To install the needed python modules you can use pip

pip3 install tabulate biopython cgecore cgelib

For more information visit the respective website

https://bitbucket.org/astanin/python-tabulate
https://biopython.org
https://bitbucket.org/genomicepidemiology/cge_core_module
https://bitbucket.org/genomicepidemiology/cgelib

BLAST (optional)

If you don't want to specify the path of blastn every time you run ResFinder, make sure that blastn is in you PATH or set the environment variable specified in the "Environment Variables Table" in this README.

Blastn can be obtained from:

ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/
https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/

# Example of how to set the environment variable in the bash shell. Remember this is only temporary, if you want it set every time you log in you need to add this line to for example your .bashrc file.
export CGE_BLASTN="/path/to/some/dir/blastn"

KMA (optional)

If you don't want to specify the path of KMA every time you run ResFinder, make sure that KMA is in you PATH or set the environment variable specified in the "Environment Variables Table" in this README.

# Go to wanted location for kma
cd /path/to/some/dir
# Clone version 1.3.25. You can also clone latest version by omitting --branch 1.3.23 or change the version number.
git clone --depth 1 --branch 1.3.23 https://bitbucket.org/genomicepidemiology/kma.git
# Compile KMA
cd kma && make
# Example of how to set the environment variable in the bash shell. Remember this is only temporary, if you want it set every time you log in you need to add this line to for example your .bashrc file.
export CGE_KMA="/path/to/some/dir/kma/kma"

Databases

If you don't want to specify the path to the databases every time you run ResFinder, you need to set the environment variable specified in the "Environment Variables Table" in this README.

ResFinder database

# Go to wanted location for the ResFinder database.
cd /path/to/some/dir/
git clone https://[e-mail]/genomicepidemiology/resfinder_db.git
# Example of how to set the environment variable in the bash shell. Remember this is only temporary, if you want it set every time you log in you need to add this line to for example your .bashrc file.
export CGE_RESFINDER_RESGENE_DB="/path/to/some/dir/resfinder_db"

PointFinder database

# Go to wanted location for the PointFinder database.
cd /path/to/some/dir/
git clone https://[e-mail]/genomicepidemiology/pointfinder_db.git
# Example of how to set the environment variable in the bash shell. Remember this is only temporary, if you want it set every time you log in you need to add this line to for example your .bashrc file.
export CGE_RESFINDER_RESPOINT_DB="/path/to/some/dir/pointfinder_db"

Indexing databases with KMA

If you have KMA installed you either need to have the kma_index in your PATH or you need to provide the path to kma_index to INSTALL.py

NOTE: The documentation given here describes the procedure for the ResFinder database, but the procedure is identical for the PointFinder database. PointFinder database documentation: [https://bitbucket.org/genomicepidemiology/pointfinder_db]

a) Run INSTALL.py in non_interactive mode

# Go to the database directory
cd path/to/resfinder_db
python3 INSTALL.py /path/to/some/dir/kma/kma_index non_interactive

The path to kma_index can be omitted if it exists in PATH or if the script should attempt to do an automatic temporary installation of KMA.

b) Run INSTALL.py in interactive mode

# Go to the database directory
cd path/to/resfinder_db
python3 INSTALL.py

If kma_index was found in your path a lot of indexing information will be printed to your terminal, and will end with the word "done".

If kma_index wasn't found you will recieve the following output:

KMA index program, kma_index, does not exist or is not executable
Please input path to executable kma_index program or choose one of the options below:
	1. Install KMA using make, index db, then remove KMA.
	2. Exit

You can now write the path to kma_index and finish with or you can enter "1" or "2" and finish with .

If "1" is chosen, the script will attempt to install kma in your systems default temporary location. If the installation is successful it will proceed to index your database, when finished it will delete the kma installation again.

Install ResFinder with Docker

If you would like to build a docker image with ResFinder, make sure you have cloned the ResFinder directory as well as installed and indexed the databases: db_pointfinder and db_resfinder. Then run the following commands:

# Go to ResFinder directory
cd path/to/resfinder
# Build docker image with name resfinder
docker build -t resfinder .

When running the docker make sure to mount the db_resfinder and the db_pointfinder with the flag -v, as shown in the examples below.

You can test the installation by running the docker with the test files:

cd path/to/resfinder/
mkdir results

# Run with raw data (this command mounts the results to the local directory "results")
docker run --rm -it -v $(pwd)/db_resfinder/:/usr/src/db_resfinder -v $(pwd)/results/:/usr/src/results resfinder -ifq /usr/src/tests/data/test_isolate_01_1.fq /usr/src/tests/data/test_isolate_01_2.fq -acq -db_res /usr/src/db_resfinder -o /usr/src/results

# Run with assembled data (this command mounts the results to the local directory "results")
docker run --rm -it -v $(pwd)/db_resfinder/:/usr/src/db_resfinder  -v $(pwd)/results/:/usr/src/results resfinder -ifa /usr/src/tests/data/test_isolate_01.fa -acq -db_res /usr/src/db_resfinder -o /usr/src/results

Test ResFinder intallation

(This will not function with the docker installation.) If you did not install BLAST, test 1 and 3 will fail. If you did not install KMA, test 2 and 4 will fail. The 4 tests will in total take approximately take 5-60 seconds, depending on your system.

Test if no paths needs to be specified

Requirements

• blastn/kma is in your PATH or specified via environment variables.
• ResFinder database and PointFinder database paths are specified via environment variables.

cd /path/to/some/dir/resfinder

# Run tests
python3 tests/functional_tests.py

# Output from successful tests
....
----------------------------------------------------------------------
Ran 4 tests in 8.263s

OK

Test if paths needs to be specified

If blastn/kma is not in your PATH or set as environment variables or the paths to the ResFinder and PointFinder databases is not set in environment variables, you need to specify the paths to these in the test script.

# Go to the directoy in which you installed the ResFinder tool
cd /path/to/some/dir/resfinder

# To see the flags you need to set for the test run:
python3 tests/functional_tests.py -res_help

# Which outputs:
usage: functional_tests.py [-res_help] [-db_res DB_PATH_RES] [-b BLAST_PATH]
                           [-k KMA_PATH] [-db_point DB_PATH_POINT]

Options:
  -res_help, --resfinder_help
  -db_res DB_PATH_RES, --db_path_res DB_PATH_RES
                        Path to the databases for ResFinder
  -b BLAST_PATH, --blastPath BLAST_PATH
                        Path to blastn
  -k KMA_PATH, --kmaPath KMA_PATH
                        Path to KMA
  -db_point DB_PATH_POINT, --db_path_point DB_PATH_POINT
                        Path to the databases for PointFinder

# Run tests with the options you need to set. For example:
python3 tests/functional_tests.py -k /path/to/some/dir/kma/kma

# Output from successful tests
....
----------------------------------------------------------------------
Ran 4 tests in 8.263s

OK

Test data

Test data can be found in the sub-dierectory /tests/data

Usage

You can run resfinder command line using python3.

NOTE: Species should be entered with their full scientific names (e.g. "escherichia coli"), using quotation marks, not case sensitive. An attempt has been made to capture some deviations like "ecoli" and "e.coli", but it is far from all deviations that will be captured.

# Example of running resfinder
python3 run_resfinder.py -o path/to/outdir -s "Escherichia coli" -l 0.6 -t 0.8 --acquired --point -ifq test_isolate_01_*

# The program can be invoked with the -h option
usage: run_resfinder.py [-h] [-ifa INPUTFASTA]
                        [-ifq INPUTFASTQ [INPUTFASTQ ...]] [-scripts SCRIPTS]
                        [-o OUT_PATH] [-b BLAST_PATH] [-k KMA_PATH]
                        [-s SPECIES] [-l MIN_COV] [-t THRESHOLD]
                        [-db_res DB_PATH_RES] [-db_res_kma DB_PATH_RES_KMA]
                        [-d DATABASES] [-acq] [-c] [-db_point DB_PATH_POINT]
                        [-g SPECIFIC_GENE [SPECIFIC_GENE ...]] [-u]

optional arguments:
  -h, --help            show this help message and exit
  -ifa INPUTFASTA, --inputfasta INPUTFASTA
                        Input fasta file.
  -ifq INPUTFASTQ [INPUTFASTQ ...], --inputfastq INPUTFASTQ [INPUTFASTQ ...]
                        Input fastq file(s). Assumed to be single-end fastq if
                        only one file is provided, and assumed to be paired-
                        end data if two files are provided.
  -o OUT_PATH, --outputPath OUT_PATH
                        All output will be stored in this directory.
  -b BLAST_PATH, --blastPath BLAST_PATH
                        Path to blastn
  -k KMA_PATH, --kmaPath KMA_PATH
                        Path to kma
  -s SPECIES, --species SPECIES
                        Species in the sample
						Available species: Campylobacter, Campylobacter jejuni, Campylobacter coli,
						Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Helicobacter pylori,
						Klebsiella, Mycobacterium tuberculosis, Neisseria gonorrhoeae,
						Plasmodium falciparum, Salmonella, Salmonella enterica, Staphylococcus aureus
						-s "Other" can be used for metagenomic samples or samples with unknown species.
  -db_res DB_PATH_RES, --db_path_res DB_PATH_RES
                        Path to the databases for ResFinder
  -db_res_kma DB_PATH_RES_KMA, --db_path_res_kma DB_PATH_RES_KMA
                        Path to the ResFinder databases indexed with KMA.
                        Defaults to the 'kma_indexing' directory inside the
                        given database directory.
  -d DATABASES, --databases DATABASES
                        Databases chosen to search in - if none is specified
                        all is used
  -acq, --acquired      Run resfinder for acquired resistance genes
	-l MIN_COV, --min_cov MIN_COV
	                      Minimum (breadth-of) coverage of ResFinder
						  Valid interval: 0.00-1.00
  -t THRESHOLD, --threshold THRESHOLD
											  Threshold for identity of ResFinder
											  Valid interval: 0.00-1.00
  -c, --point           Run pointfinder for chromosomal mutations
  -db_point DB_PATH_POINT, --db_path_point DB_PATH_POINT
                        Path to the databases for PointFinder
  -g SPECIFIC_GENE [SPECIFIC_GENE ...]
                        Specify genes existing in the database to search for -
                        if none is specified all genes are included in the
                        search.
  -u, --unknown_mut     Show all mutations found even if in unknown to the
                        resistance database
	-l_p MIN_COV_POINT, --min_cov_point MIN_COV_POINT
	                      Minimum (breadth-of) coverage of Pointfinder. If None
	                      is selected, the minimum coverage of ResFinder will be
	                      used.
	-t_p THRESHOLD_POINT, --threshold_point THRESHOLD_POINT
											  Threshold for identity of Pointfinder. If None is
											  selected, the minimum coverage of ResFinder will be
											  used.

Environment Variables

Environment variables recognized by ResFinder, the flag they replace and the default value for the flag. Provided commandline flags will always take precedence. Set environment variables takes precedence over default flag values.

Additional Environment variables can be added by appending entries to the table below. The 'Flag' entry in the table must be the double dash flag recognized by ResFinder. The 'Default Value' entry is just for information.

Environment Variables Table

Environment Variabel	Flag	Default Value
CGE_KMA	kmaPath	kma
CGE_BLASTN	blastPath	blastn
CGE_RESFINDER_RESGENE_DB	db_path_res	db_resfinder
CGE_RESFINDER_RESPOINT_DB	db_path_point	db_pointfinder
CGE_RESFINDER_GENE_COV	min_cov	0.60
CGE_RESFINDER_GENE_ID	threshold	0.80
CGE_RESFINDER_POINT_COV	min_cov_point	0.60
CGE_RESFINDER_POINT_ID	threshold_point	0.80

Species Abbreviations

ResFinder understands the species abbreviations listed in the Species Abbreviations Table. Additional species abbreviations can be added by appending entries to the table below. Important! All entries should be kept in lower case, although at runtime the ResFinder species flag will be case insensitive.

Species Abbreviations Table

Species	Abbreviation
campylobacter jejuni	c. jejuni
campylobacter jejuni	c.jejuni
campylobacter jejuni	c jejuni
campylobacter jejuni	cjejuni
campylobacter coli	c. coli
campylobacter coli	c.coli
campylobacter coli	c coli
campylobacter coli	ccoli
escherichia coli	e. coli
escherichia coli	e.coli
escherichia coli	e coli
escherichia coli	ecoli
salmonella enterica	s. enterica
salmonella enterica	s.enterica
salmonella enterica	s enterica
salmonella enterica	senterica

Web-server

A webserver implementing the methods is available at the CGE website and can be found here: https://cge.cbs.dtu.dk/services/ResFinder/

Citation

When using the method please cite:

ResFinder 4.0 for predictions of phenotypes from genotypes.
Bortolaia V, Kaas RS, Ruppe E, Roberts MC, Schwarz S, Cattoir V, Philippon A, Allesoe RL, Rebelo AR, Florensa AR, Fagelhauer L, Chakraborty T, Neumann B, Werner G, Bender JK, Stingl K, Nguyen M, Coppens J, Xavier BB, Malhotra-Kumar S, Westh H, Pinholt M, Anjum MF, Duggett NA, Kempf I, Nyk�senoja S, Olkkola S, Wieczorek K, Amaro A, Clemente L, Mossong J, Losch S, Ragimbeau C, Lund O, Aarestrup FM. Journal of Antimicrobial Chemotherapy. 2020 Aug 11.
PMID: 32780112 doi: 10.1093/jac/dkaa345
[Epub ahead of print]

References

1. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. BLAST+: architecture and applications. BMC Bioinformatics 2009; 10:421.
2. Clausen PTLC, Aarestrup FM, Lund O. Rapid and precise alignment of raw reads against redundant databases with KMA. BMC Bioinformatics 2018; 19:307.

License

Licensed under the Apache License, Version 2.0 (the "License"); you may not use this file except in compliance with the License. You may obtain a copy of the License at

http://www.apache.org/licenses/LICENSE-2.0

Unless required by applicable law or agreed to in writing, software distributed under the License is distributed on an "AS IS" BASIS, WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied. See the License for the specific language governing permissions and limitations under the License.