WARNING - AMRfíor is now no longer supported and will not receive any further updates. Please use the newer GeneFíor (https://github.com/NickJD/GeneFior) toolkit for your AMR (and non-AMR) gene detection needs!!!

AMRfíor (pronounced AMR "feer", sounds like beer)

This toolkit utilises a combined approach that uses BLAST, BWA, Bowtie2, DIAMOND, and Minimap2 to search DNA and protein sequences against AMR databases (DNA and AA) such as CARD/RGI and ResFinder.

Requirements:

- python >=3.10
- samtools >=1.19.2
- blast >=2.17.0
- diamond >=2.1.13
- bowtie2 >=2.5.4
- bwa >=0.7.19
- minimap2 >=2.30
- seqtk >=1.4

Installation:

AMRfíor is available via bioconda. To install, use the following command:

conda install -c bioconda amrfior

AMRfíor is also available via pip, but bioconda is recommended to ensure all dependencies are correctly installed.

pip install amrfior

Menu for AMRfíor (AMRfíor or amrfíor):

BLASTn and BLASTx are disabled by default due to their slow speed, but can be enabled if desired. CARD and resfinder databases are used by default, but user-provided databases can also be specified. The NCBI AMR database is also available as an option. All 3 databases are prepackaged and formatted as part of the bioconda installation of AMRfíor.

AMRfíor v0.5.1 - The Multi-Tool AMR Gene Detection Toolkit.

options:
  -h, --help            show this help message and exit

Required selection:
  -i INPUT, --input INPUT
                        Input FASTA/FASTAQ file(s) with sequences to analyse - Separate FASTQ R1 and R2 with a comma for Paired-FASTQ or single file path for Single-FASTA - .gz files
                        accepted
  -st {Single-FASTA,Paired-FASTQ}, --sequence-type {Single-FASTA,Paired-FASTQ}
                        Specify the input Sequence Type: Single-FASTA or Paired-FASTQ (R1+R2) - Will convert Paired-FASTQ to single combined FASTA for BLAST and DIAMOND analyses (SLOW)
  -o OUTPUT, --output OUTPUT
                        Output directory for results

Output selection:
  --report-fasta {None,all,detected,detected-all}
                        Specify whether to output sequences that "mapped" to genes."all" should only be used for deep investigation/debugging."detected" will report the reads that passed
                        detection thresholds for each detected gene."detected-all" will report all reads for each detected gene. (default: None)

Tool selection:
  --tools {blastn,blastx,diamond,bowtie2,bwa,minimap2,all} [{blastn,blastx,diamond,bowtie2,bwa,minimap2,all} ...]
                        Specify which tools to run - "all" will run all tools (default: all except blastx/n as it is very slow!!)

Database selection:
  --databases {resfinder,card,ncbi,user-provided} [{resfinder,card,ncbi,user-provided} ...]
                        Specify which AMR gene databases to use (default: resfinder and card) -If "user-provided" is selected, please ensure the path contains the appropriate databases
                        set up as per the documentation and specify the path with --user-db-path.
  --user-db-path USER_DB_PATH
                        Path to the directory containing user-provided databases (required if --databases includes "user-provided")

Query threshold Parameters:
  --q-min-cov QUERY_MIN_COVERAGE, --query-min-coverage QUERY_MIN_COVERAGE
                        Minimum coverage threshold in percent (default: 40.0)

Gene Detection Parameters:
  --d-min-cov DETECTION_MIN_COVERAGE, --detection-min-coverage DETECTION_MIN_COVERAGE
                        Minimum coverage threshold in percent (default: 80.0)
  --d-min-id DETECTION_MIN_IDENTITY, --detection-min-identity DETECTION_MIN_IDENTITY
                        Minimum identity threshold in percent (default: 80.0)
  --d-min-base-depth DETECTION_MIN_BASE_DEPTH, --detection-min-base-depth DETECTION_MIN_BASE_DEPTH
                        Minimum average base depth for detection - calculated against regions of the detected gene with at least one read hit (default: 1.0)
  --d-min-reads DETECTION_MIN_NUM_READS, --detection-min-num-reads DETECTION_MIN_NUM_READS
                        Minimum number of reads required for detection (default: 1)

Mode Selection:
  --dna-only            Run only DNA-based tools
  --protein-only        Run only protein-based tools
  --sensitivity {default,conservative,sensitive,very-sensitive}
                        Preset sensitivity levels - default means each tool uses its own default settings and very-sensitive applies DIAMONDs --ultra-sensitive and Bowtie2s --very-
                        sensitive-local presets

Tool-Specific Parameters:
  --minimap2-preset {sr,map-ont,map-pb,map-hifi}
                        Minimap2 preset: sr=short reads, map-ont=Oxford Nanopore, map-pb=PacBio, map-hifi=PacBio HiFi (default: sr)

Runtime Parameters:
  -t THREADS, --threads THREADS
                        Number of threads to use (default: 4)
  -tmp TEMP_DIRECTORY, --temp-directory TEMP_DIRECTORY
                        Path to temporary to place input FASTA/Q file(s) for faster IO during BLAST - Path will also be used for all temporary files (default: system temp directory)
  --no_cleanup
  --verbose

Miscellaneous Parameters:
  -v, --version         Show program version and exit

Examples:
  # Basic usage with default tools (runs DNA & protein tools)
  AMRfior -i reads.fasta -st Single-FASTA -o results/

  # Select specific tools and output detected FASTA sequences
  AMRfior -i reads.fasta -st Single-FASTA -o results/     --tools diamond bowtie2     --report_fasta detected

  # Custom thresholds, paired-fastq input, threads and dna-only mode
  AMRfior -i reads_R1.fastq,reads_R2.fastq -st Paired-FASTQ -o results/     -t 16 --d-min-cov 90 --d-min-id 85     --dna-only

Menu for AMRfíor-Recompute (AMRfíor-Recompute or amrfíor-recompute):

AMRfíor-Recompute is used to recalculate detection statistics from existing sequence search outputs with different thresholds without needing to rerun the entire analysis.

AMRfíor v0.5.1 - AMRfíor-Recompute: Recalculate detection statistics from existing sequence search outputs

options:
  -h, --help            show this help message and exit
  -i INPUT, --input INPUT
                        Input directory containing AMRfíor results (with
                        raw_outputs/ subdirectory)
  -o OUTPUT, --output OUTPUT
                        Output directory for recomputed results
  --tools {blastn,blastx,diamond,bowtie2,bwa,minimap2,all} [{blastn,blastx,diamond,bowtie2,bwa,minimap2,all} ...]
                        Specify which tools to recompute - "all" will
                        recompute for all detected tools (default: all)

Query threshold Parameters:
  --q-min-cov QUERY_MIN_COVERAGE, --query-min-coverage QUERY_MIN_COVERAGE
                        Minimum coverage threshold in percent (default: 40.0)

Gene Detection Parameters:
  --d-min-cov DETECTION_MIN_COVERAGE, --detection-min-coverage DETECTION_MIN_COVERAGE
                        Minimum coverage threshold in percent (default: 80.0)
  --d-min-id DETECTION_MIN_IDENTITY, --detection-min-identity DETECTION_MIN_IDENTITY
                        Minimum identity threshold in percent (default: 80.0)
  --d-min-base-depth DETECTION_MIN_BASE_DEPTH, --detection-min-base-depth DETECTION_MIN_BASE_DEPTH
                        Minimum average base depth for detection - calculated
                        against regions of the detected gene with at least one
                        read hit (default: 1.0)
  --d-min-reads DETECTION_MIN_NUM_READS, --detection-min-num-reads DETECTION_MIN_NUM_READS
                        Minimum number of reads required for detection
                        (default: 1)

Output Parameterts:
  --report-fasta {None,all,detected,detected-all}
                        Specify whether to output sequences that "mapped" to
                        genes."all" should only be used for deep
                        investigation/debugging."detected" will report the
                        reads that passed detection thresholds for each
                        detected gene."detected-all" will report all reads for
                        each detected gene. (default: None)
  --query-fasta QUERY_FASTA
                        Specify the original query FASTA/FASTQ file used for
                        alignment (required for reporting mapped sequences for
                        BLAST/DIAMOND).

Miscellaneous Parameters:
  -v, --version         Show program version and exit

Examples:
  # Recompute with different thresholds
  AMRfior-recompute -i original_results/ -o recomputed_90_90/ \
    --d-min-cov 90 --d-min-id 90

  # More stringent depth requirement
  AMRfior-recompute -i original_results/ -o high_depth/ \
    --d-min-base-depth 5.0 --d-min-reads 10

Menu for AMRfíor-Gene-Stats (AMRfíor-Gene-Stats or amrfíor-gene-stats):

AMRfíor-Gene-Stats is used to generate summary statistics and visualizations from AMRfíor results.

AMRfíor v0.5.1 - AMRfíor-Gene-Stats: Generate detailed coverage visualisations for AMR genes

options:
  -h, --help            show this help message and exit
  -i INPUT, --input INPUT
                        Input directory containing AMRfíor results
  -o OUTPUT, --output OUTPUT
                        Output directory for visualisation reports
  -g GENES, --genes GENES
                        Comma-separated gene names (FULL NAMES) or path to file with gene names (one per line)
  --databases {resfinder,card,ncbi} [{resfinder,card,ncbi} ...]
                        Database(s) to interrogate
  --tools {blastn,blastx,diamond,bowtie2,bwa,minimap2,all} [{blastn,blastx,diamond,bowtie2,bwa,minimap2,all} ...]
                        Tool(s) to interrogate
  --ref-fasta REF_FASTA
                        NOT IMPLEMENTED YET - Reference FASTA file for variant calling (optional)
  --query-fasta QUERY_FASTA
                        NOT IMPLEMENTED YET - Query FASTA file (your input reads) for BLAST base-level analysis (optional)

Examples:
  # Visualise specific genes (FULL NAMES) from all tools
  AMRfior-gene-stats -i results/ -o vis/ \
    -g "sul1_2_U12338,tet(W)|ARO:3000194" \
    --databases resfinder card \
    --tools diamond bowtie2 bwa

  # Visualise from gene (FULL NAMES) list file with reference
  AMRfior-gene-stats -i results/ -o vis/ \
    -g genes_of_interest.txt \
    --databases resfinder \
    --tools blastn diamond 

Database Setup: See /src/AMRfior/databases/ for details on setting up user-provided databases.

AMRfíor includes an automated script in the Databases directory to automate the setup of user-provided databases.