Pharokka

https://pharokka.readthedocs.io/en/latest/
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Pharokka is a fast phage annotation pipeline that uses gene prediction and functional annotations to...
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### Install Miniforge on Linux x86_64

Source: https://pharokka.readthedocs.io/en/latest/install

Downloads the Miniforge installer script for Linux x86_64 systems. This is part of the beginner's guide to setting up Conda.

```bash
wget https://github.com/conda-forge/miniforge/releases/download/24.9.2-0/Miniforge3-24.9.2-0-Linux-x86_64.sh

```

--------------------------------

### Execute Miniforge installer script

Source: https://pharokka.readthedocs.io/en/latest/install

Runs the downloaded Miniforge installer script to install Conda. Follow the on-screen prompts for completion.

```bash
sh Miniforge3-24.9.2-0-Linux-x86_64.sh

```

--------------------------------

### Install Miniforge on Mac Intel

Source: https://pharokka.readthedocs.io/en/latest/install

Downloads the Miniforge installer script for Mac Intel systems. This is part of the beginner's guide to setting up Conda.

```bash
wget https://github.com/conda-forge/miniforge/releases/download/24.9.2-0/Miniforge3-24.9.2-0-MacOSX-x86_64.sh

```

--------------------------------

### Install Pharokka dependencies and from source

Source: https://pharokka.readthedocs.io/en/latest/install

Installs Pharokka dependencies using Conda from environment.yml, then installs Pharokka from source using pip.

```bash
conda env create -f environment.yml
conda activate pharokka_env
# assuming you are in the pharokka directory 
# installs pharokka from source
pip install -e .
pharokka.py --help

```

--------------------------------

### Install Pharokka using Conda

Source: https://pharokka.readthedocs.io/en/latest/install

Recommended method for easy installation of Pharokka and its dependencies.

```bash
conda install -c bioconda pharokka

```

--------------------------------

### Install Pharokka from source using Git and Pip

Source: https://pharokka.readthedocs.io/en/latest/install

Installs the development version of Pharokka directly from GitHub. Requires manual installation of dependencies listed in environment.yml.

```bash
git clone https://github.com/gbouras13/pharokka.git
cd pharokka
pip install -e .
pharokka.py --help

```

--------------------------------

### Example Command for Creating Custom HMMs

Source: https://pharokka.readthedocs.io/en/latest/custom

This command demonstrates how to use `create_custom_hmm.py` with a specific input directory (`my_tail_msa`), output directory (`tail_hmms`), and prefix (`tail`).

```bash
create_custom_hmm.py -i my_tail_msa -o tail_hmms -p tail
```

--------------------------------

### Install Miniforge on Mac M1/M2/M3/M4

Source: https://pharokka.readthedocs.io/en/latest/install

Downloads the Miniforge installer script for Mac ARM64 systems (M1/M2/M3/M4). This is part of the beginner's guide to setting up Conda.

```bash
wget https://github.com/conda-forge/miniforge/releases/download/24.9.2-0/Miniforge3-24.9.2-0-MacOSX-arm64.sh

```

--------------------------------

### Install Pharokka Python components using Pip

Source: https://pharokka.readthedocs.io/en/latest/install

Installs only the Python components of Pharokka. Non-Python dependencies must be installed manually.

```bash
pip install pharokka

```

--------------------------------

### Create and activate Conda environment for Pharokka

Source: https://pharokka.readthedocs.io/en/latest/install

Creates a new Conda environment named 'pharokkaENV', installs Pharokka into it, and activates the environment. Also shows commands to check database and Pharokka help.

```bash
conda create -n pharokkaENV pharokka
conda activate pharokkaENV
install_databases.py -h
pharokka.py -h

```

--------------------------------

### Add Conda channels for Pharokka installation

Source: https://pharokka.readthedocs.io/en/latest/install

Configures Conda to use the 'defaults', 'bioconda', and 'conda-forge' channels, which are necessary for installing Pharokka.

```bash
conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge

```

--------------------------------

### Create and activate Conda environment for Pharokka on Mac Apple Silicon

Source: https://pharokka.readthedocs.io/en/latest/install

Creates a new Conda environment for Pharokka on Mac with Apple Silicon, installs Pharokka, and activates the environment. Also shows commands to check database and Pharokka help.

```bash
conda create --platform osx-64 -n pharokkaENV pharokka
conda activate pharokkaENV
install_databases.py -h
pharokka.py -h

```

--------------------------------

### Example MSA Input Format

Source: https://pharokka.readthedocs.io/en/latest/custom

The `create_custom_hmm.py` script expects FASTA formatted Multiple Sequence Alignments (MSAs) as input. Each gene name serves as the custom annotation for its corresponding HMM.

```fasta
>gene1
--------------------MERCMYDLSHFSFMTGRIGQLQTLTCIPVVSGDSFSVNFQGVFRLSPLRRNMVIDCMVDLFAFYVPYRHVYG-----DDWENFMLQGTDETVTFGTAD-FTTN-AVEYLGSQ-----R-TTGVAPLWRLASYNQIWNRYFRVPSDTSAILSNTALE---A-GVNKNKWGRYCARPKVIWSTGVDTTTD--AAD--REVTVTANQLDITDLDAVKGRYASEQRRDWFG-QRYNDILKNQFGGGAGTDADERPTLIMRKQHFLSGYDVDGTGDATLGQYSGKSAAVCDLQIPRKWFPEHGSL-WIMALLRFPTIHEEEIHYLFKKSQPTYDEISGDPDIWERKAPIEHQVQDFFTESTDTTSLGFMPYGQWYRYHPNVVHNQFTEVTGFSFVSA-RPT-----SKDTARYIQDDEYTPTFQTTSLGHWQSQARVDVEAVRPIPGPLKSIFAGV------------
>gene2
MSKSSGYATTSKPQEVVTRDIERKPYDLSHWSFKTGHIGRLQTFSVIPVVAGDSIELNMSAVLRLSPLRHFMYLDAVVDLFAFYVPHRHVYG-----SNWTAFLKAGVDEGSTLGTTT-FTGSNRPECLGVY-----FAAGQVLPSWSVHPYTMIWNNYFRDPQVDADAKTDATYLIGLTYPDPILKYGLPVCHLKRSWNTGVASGLS--TAD--YSLALSGGEVDLYQMSQLKGRLQTEQARDWFAFARYRDILKYTWDSEVNIDADQRPELIMRSTAWLSGKDVDGTDDATLGSYTGKSTGIFNLSFPSRFFAEHGSI-WIMGVVRFPPIVEAEAGYLNSKSEPTYKEISGDPSIIKHEPPISLNANEQFQG-ASSVDLGKIPYGQWYRESINMLSADYLEVSGHPFLLKSSIT-----SRATGVYVVPSQYDTVFSDTLMGHWNSQAFVDVRAKRFIPAPEDSIFAGTI----------- 
>gene3
----------------MKGTDTRCLYDLDHWSHVSGNIGSLQTLSAIPVVAGDSMELNFTSLFRLSPLRRNLYLDAMVDLFAFYVPYRHVYG-----DTWIDFIKEGYDESQTLGTYT-IAAGEFINCTGAY-----LESQAVIPKWSIASYTRIWNRYFRHPTDSNEKDDDDLI----TASSGNQIYGYNCCHMKNIWSTGVDSELT--DDD--HKVDVTASKVDLLEIIQKKARFKTERQREWFG-QRYTDILDSVWGSNVNIDADERPELIMRTSTWLSGYDVDGTTETNIGTFSGKAFTTARLQFPMKYFNEHGTI-YIVALVRFPTIHAFERHYLFGKSEPTYKEIAGDPNVIRNEPPQAINPTDYIEN-ATATDVGLQPYAQWYRTHPSYTSLGFSNLDGHPFLQK-IIA-----NTDDAVYVDSNDYDEIFQTQQLQQWQSQGYVGLKAKRNIPDPRKSIFAGVK-----------
```

--------------------------------

### Install Pharokka on MacOS with Apple Silicon (M1/M2/M3)

Source: https://pharokka.readthedocs.io/en/latest/install

Specific Conda installation command for MacOS systems with Apple Silicon to ensure compatibility.

```bash
conda create --platform osx-64 --name pharokkaENV -c bioconda pharokka
conda activate pharokkaENV

```

--------------------------------

### Example Locus Tags File Content

Source: https://pharokka.readthedocs.io/en/latest/plotting

Content of a text file used with `--label_ids` to specify which locus tags should be labeled on the plot.

```text
EHOONAYF_CDS_0007
EHOONAYF_CDS_0009
EHOONAYF_CDS_0023

```

--------------------------------

### Pass custom arguments to MINced

Source: https://pharokka.readthedocs.io/en/latest/run

Provide custom arguments to the MINced tool. Ensure arguments do not start with a hyphen and are enclosed in quotes if they contain spaces.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --minced_args "minNR 2 -minRL 21"
```

--------------------------------

### Run Pharokka with automatic DNA reorientation

Source: https://pharokka.readthedocs.io/en/latest/run

Automatically reorient the phage sequence to start with the large terminase subunit using the `--dnaapler` flag. The reoriented FASTA file will be named with a `_dnaapler_reoriented` suffix.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --dnaapler
```

--------------------------------

### Manually reorient phage using terminase coordinates (positive strand)

Source: https://pharokka.readthedocs.io/en/latest/run

Manually reorient the phage sequence to begin at a specific coordinate, typically the start of the large terminase subunit on the positive strand. Requires the input to be a single contig.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --terminase --terminase_start 9503 --terminase_strand pos
```

--------------------------------

### Basic Pharokka Run

Source: https://pharokka.readthedocs.io/en/latest/run

This is the most basic command to run pharokka. Ensure you have an input FASTA file and specify an output folder and the number of threads.

```bash
pharokka.py -i <fasta file> -o <output folder> -t <threads> -p <prefix>
```

--------------------------------

### create_custom_hmm.py Script Usage

Source: https://pharokka.readthedocs.io/en/latest/custom

This displays the command-line options for the `create_custom_hmm.py` script, including arguments for input directory, output directory, prefix, and force overwrite.

```bash
usage: create_custom_hmm.py [-h] [-i INDIR] [-o OUTDIR] [-p PREFIX] [-f] [-V]

create_custom_hmm.py: Creates HMMs from FASTA formatted MSAs with PyHMMER for use with Pharokka v1.4.0 and higher.

options:
  -h, --help            show this help message and exit
  -i INDIR, --indir INDIR
                        Input directory containing FASTA formatted Multiple Sequence Alignments.
  -o OUTDIR, --outdir OUTDIR
                        Output directory to store HMM profiles.
  -p PREFIX, --prefix PREFIX
                        Prefix used to name HMMs. The relevant file be 'prefix'.h3m
  -f, --force           Overwrites the output directory.
  -V, --version         Print pharokka Version

```

--------------------------------

### Run Pharokka in meta mode with split output

Source: https://pharokka.readthedocs.io/en/latest/run

Enable meta mode and activate split mode for output. This generates separate directories for FASTA, GenBank, and GFF files for each input contig.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  -m -s
```

--------------------------------

### Pharokka Command-Line Help

Source: https://pharokka.readthedocs.io/en/latest/run

Displays the full help message for the Pharokka command-line interface, listing all available options and their descriptions. This is useful for understanding the program's capabilities and parameters.

```bash
usage: pharokka.py [-h] [-i INFILE] [-o OUTDIR] [-d DATABASE] [-t THREADS] [-f] [-p PREFIX] [-l LOCUSTAG] [-g GENE_PREDICTOR] [-m] [-s] [-c CODING_TABLE] [-e EVALUE] [--fast] [--mmseqs2_only] [--meta_hmm] [--dnaapler] [--custom_hmm CUSTOM_HMM] [--genbank] [--terminase] [--terminase_strand TERMINASE_STRAND] [--terminase_start TERMINASE_START] [--skip_extra_annotations] [--skip_mash] [--minced_args MINCED_ARGS] [--mash_distance MASH_DISTANCE] [-V] [--citation]
pharokka: fast phage annotation program
options: -h, --help show this help message and exit -i INFILE, --infile INFILE Input genome file in fasta format. -o OUTDIR, --outdir OUTDIR Directory to write the output to. -d DATABASE, --database DATABASE Database directory. If the databases have been installed in the default directory, this is not required. Otherwise specify the path. -t THREADS, --threads THREADS Number of threads. Defaults to 1. -f, --force Overwrites the output directory. -p PREFIX, --prefix PREFIX Prefix for output files. This is not required. -l LOCUSTAG, --locustag LOCUSTAG User specified locus tag for the gff/gbk files. This is not required. A random locus tag will be generated instead. -g GENE_PREDICTOR, --gene_predictor GENE_PREDICTOR User specified gene predictor. Use "-g phanotate" or "-g prodigal" or "-g prodigal-gv" or "-g genbank". Defaults to phanotate (not required unless prodigal is desired). -m, --meta meta mode for metavirome input samples -s, --split split mode for metavirome samples. -m must also be specified. Will output separate split FASTA, gff and genbank files for each input contig. -c CODING_TABLE, --coding_table CODING_TABLE translation table for prodigal. Defaults to 11. -e EVALUE, --evalue EVALUE E-value threshold for MMseqs2 database PHROGs, VFDB and CARD and PyHMMER PHROGs database search. Defaults to 1E-05. --fast, --hmm_only Runs PyHMMER (HMMs) with PHROGs only, not MMseqs2 with PHROGs, CARD or VFDB. Designed for phage isolates, will not likely be faster for large metagenomes. --mmseqs2_only Runs MMseqs2 with PHROGs, CARD and VFDB only (same as Pharokka v1.3.2 and prior). Default in meta mode. --meta_hmm Overrides --mmseqs2_only in meta mode. Will run both MMseqs2 and PyHMMER. --dnaapler Runs dnaapler to automatically re-orient all contigs to begin with terminase large subunit if found. Recommended over using '--terminase'. --custom_hmm CUSTOM_HMM Run pharokka with a custom HMM profile database suffixed .h3m. Please use create this with the create_custom_hmm.py script. --genbank Flag denoting that -i/--input is a genbank file instead of the usual FASTA file. The CDS calls in this file will be preserved and re-annotated. --terminase Runs terminase large subunit re-orientation mode. Single genome input only and requires --terminase_strand and --terminase_start to be specified. --terminase_strand TERMINASE_STRAND Strand of terminase large subunit. Must be "pos" or "neg". --terminase_start TERMINASE_START Start coordinate of the terminase large subunit. --skip_extra_annotations Skips tRNAscan-se, MINced and Aragorn. --skip_mash Skips running mash to find the closest match for each contig in INPHARED. --minced_args MINCED_ARGS extra commands to pass to MINced (please omit the leading hyphen for the first argument). You will need to use quotation marks e.g. --minced_args "minNR 2 -minRL 21" --mash_distance MASH_DISTANCE mash distance for the search against INPHARED. Defaults to 0.2. --trna_scan_model {general,bacterial} tRNAscan-SE model -V, --version Print pharokka Version --citation Print pharokka Citation 
```

--------------------------------

### Specify Database Directory

Source: https://pharokka.readthedocs.io/en/latest/run

It is recommended to specify the database directory using the `-d` flag, especially if it's not in the default location.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/databse_dir> -t <threads> -p <prefix>
```

--------------------------------

### Download and extract Pharokka databases

Source: https://pharokka.readthedocs.io/en/latest/install

Downloads the Pharokka database archive from Zenodo and extracts it. This is necessary for versions 1.4.0 and higher.

```bash
wget "https://zenodo.org/record/8276347/files/pharokka_v1.4.0_databases.tar.gz"
tar -xzf pharokka_v1.4.0_databases.tar.gz

```

--------------------------------

### Run Pharokka in meta mode with PyHMMER on PHROGs

Source: https://pharokka.readthedocs.io/en/latest/run

Enable meta mode and force the use of PyHMMER on PHROGs. Be aware that this can be time-consuming for large metagenomes.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  -m --meta_hmm
```

--------------------------------

### Run Pharokka Multiplotter

Source: https://pharokka.readthedocs.io/en/latest/plotting

Basic command to run the pharokka_multiplotter.py script with input Genbank file and output directory.

```bash
pharokka_multiplotter.py -g pharokka.gbk  -o pharokka_plots_output_directory 

```

--------------------------------

### Run Pharokka in meta mode with fast PyHMMER

Source: https://pharokka.readthedocs.io/en/latest/run

Enable meta mode and use a faster PyHMMER analysis. This option is suitable when speed is a priority in meta mode.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  -m --fast
```

--------------------------------

### Running Pharokka with a Custom HMM Database

Source: https://pharokka.readthedocs.io/en/latest/custom

Integrate your custom HMM database into the `pharokka.py` analysis by specifying the path to the generated `.h3m` file using the `--custom_hmm` parameter.

```bash
pharokka.py -i <input fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --custom_hmm tail_hmms/tail.h3m
```

--------------------------------

### Basic Plotting Command

Source: https://pharokka.readthedocs.io/en/latest/plotting

Generates a basic circular genome plot in PNG format. Requires input FASTA, a plot name, and an output directory.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory
```

--------------------------------

### Run Basic Protein Annotation

Source: https://pharokka.readthedocs.io/en/latest/proteins

This is the most basic command to run pharokka_proteins.py. It will perform MMseqs2 searches on PHROGs, CARD, and VFDB, as well as a PyHMMER search on PHROGs.

```bash
pharokka_proteins.py -i <inpu protein FASTA file> -d <database directory> -o <output folder> -t <threads>
```

--------------------------------

### Create Custom HMM Database

Source: https://pharokka.readthedocs.io/en/latest/custom

Use this script to generate HMM profiles from a directory of MSAs. The output is a `.h3m` file compatible with pharokka's `--custom_hmm` parameter.

```bash
create_custom_hmm.py -i <input directory of MSAs> -o <outdir> -p <prefix>
```

--------------------------------

### Pharokka Plotter Script Options

Source: https://pharokka.readthedocs.io/en/latest/plotting

This snippet details all available command-line options for the pharokka_plotter.py script, including input/output file specifications, plot customization, and feature labeling.

```bash
usage: pharokka_plotter.py [-h] -i INFILE [-n PLOT_NAME] [-o OUTDIR] [--gff GFF] [--genbank GENBANK] [-p PREFIX] [-t PLOT_TITLE] [-f]
                           [--label_hypotheticals] [--remove_other_features_labels] [--title_size TITLE_SIZE] [--label_size LABEL_SIZE]
                           [--interval INTERVAL] [--truncate TRUNCATE] [--dpi DPI] [--annotations ANNOTATIONS] [--label_ids LABEL_IDS]

pharokka_plotter.py: pharokka plotting function

options:
  -h, --help            show this help message and exit
  -i INFILE, --infile INFILE
                        Input genome file in FASTA format.
  -n PLOT_NAME, --plot_name PLOT_NAME
                        Output plot file name. "." suffix will be added to this automatically. 
                        Will be output in the pharokka output directory if -o is specified, or in the working directory if --gff andf --genbank are specified.
  -o OUTDIR, --outdir OUTDIR
                        Pharokka output directory.
  --gff GFF             Pharokka gff.
  --genbank GENBANK     Pharokka genbank.
  -p PREFIX, --prefix PREFIX
                        Prefix used to create pharokka output. Will default to pharokka.
  -t PLOT_TITLE, --plot_title PLOT_TITLE
                        Plot name.
  -f, --force           Overwrites the output plot file.
  --label_hypotheticals
                        Flag to label  hypothetical or unknown proteins. By default these are not labelled.
  --remove_other_features_labels
                        Flag to remove labels for tRNA/tmRNA/CRISPRs. By default these are labelled. 
                        They will still be plotted in black.
  --title_size TITLE_SIZE
                        Controls title size. Must be an integer. Defaults to 20.
  --label_size LABEL_SIZE
                        Controls annotation label size. Must be an integer. Defaults to 8.
  --interval INTERVAL   Axis tick interval. Must be an integer. Must be an integer. Defaults to 5000.
  --truncate TRUNCATE   Number of characters to include in annoation labels before truncation with ellipsis. 
                        Must be an integer. Defaults to 20.
  --dpi DPI             Resultion (dots per inch). Must be an integer. Defaults to 600.
  --annotations ANNOTATIONS
                        Controls the proporition of annotations labelled. Must be a number between 0 and 1 inclusive. 
                        0 = no annotations, 0.5 = half of the annotations, 1 = all annotations. 
                        Defaults to 1. Chosen in order of CDS size.
  --label_ids LABEL_IDS
                        Text file with list of CDS IDs (from gff file) that are guaranteed to be labelled.
```

--------------------------------

### Run Pharokka with GenBank input

Source: https://pharokka.readthedocs.io/en/latest/run

Specify a GenBank file as input using the `--genbank` flag. This bypasses Prodigal/PHANOTATE gene prediction and uses existing CDS calls from the GenBank file, recommended for manually curated data.

```bash
pharokka.py -i <GENBANK file> -o <output folder> -d <path/to/database_dir> -t <threads>  --genbank
```

--------------------------------

### Fast Mode for Small Inputs

Source: https://pharokka.readthedocs.io/en/latest/run

Use the `--fast` flag for smaller inputs like phage isolates or a few contigs. This mode only runs PyHMMER against PHROGs, skipping VFDB and CARD annotations.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --fast
```

--------------------------------

### Download Pharokka container image using Singularity

Source: https://pharokka.readthedocs.io/en/latest/install

Pulls the Pharokka Docker container image for use with Singularity. Specify the desired directory for the .sif file.

```bash
IMAGE_DIR="<the directory you want the .sif file to be in >"
# e.g. to pull into the working directory
IMAGE_DIR=$PWD
singularity pull --dir $IMAGE_DIR docker://quay.io/biocontainers/pharokka:1.7.3--pyhdfd78af_0

```

--------------------------------

### Specifying GFF and Genbank Files Directly

Source: https://pharokka.readthedocs.io/en/latest/plotting

Provides direct paths to GFF and Genbank files instead of a directory. The plot will be saved in the working directory.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot --gff pharokka.gff --genbank pharokka.gbk
```

--------------------------------

### Force Overwrite Output Directory

Source: https://pharokka.readthedocs.io/en/latest/run

Use the `-f` or `--force` flag to overwrite an existing output directory. Use with caution.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/databse_dir> -t <threads> -p <prefix>  -f
```

--------------------------------

### Run Pharokka using Singularity exec

Source: https://pharokka.readthedocs.io/en/latest/install

Executes Pharokka commands within a Singularity container. Ensure the containerImage variable points to the correct .sif file.

```bash
containerImage="$IMAGE_DIR/pharokka_1.7.3--pyhdfd78af_0.sif"
singularity exec $containerImage pharokka.py -h

```

--------------------------------

### Plotting with a Pharokka Prefix

Source: https://pharokka.readthedocs.io/en/latest/plotting

Use this command if a prefix was specified during the Pharokka analysis. It ensures correct file association for plotting.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory -p my_prefix
```

--------------------------------

### Pharokka Multiplotter Usage

Source: https://pharokka.readthedocs.io/en/latest/plotting

Displays the full usage information for the pharokka_multiplotter.py script, detailing all available options and their parameters.

```bash
usage: pharokka_multiplotter.py [-h] -g GENBANK -o OUTDIR [-f] [--label_hypotheticals] [--remove_other_features_labels]
                                [--title_size TITLE_SIZE] [--label_size LABEL_SIZE] [--interval INTERVAL] [--truncate TRUNCATE] [--dpi DPI]
                                [--annotations ANNOTATIONS] [-t PLOT_TITLE] [--label_ids LABEL_IDS]

pharokka_multiplotter.py: pharokka plotting function for muliple phages

options:
  -h, --help            show this help message and exit
  -g GENBANK, --genbank GENBANK
                        Input genbank file from Pharokka.
  -o OUTDIR, --outdir OUTDIR
                        Pharokka output directory.
  -f, --force           Overwrites the output plot file.
  --label_hypotheticals
                        Flag to label  hypothetical or unknown proteins. By default these are not labelled.
  --remove_other_features_labels
                        Flag to remove labels for tRNA/tmRNA/CRISPRs. By default these are labelled.
                        They will still be plotted in black.
  --title_size TITLE_SIZE
                        Controls title size. Must be an integer. Defaults to 20.
  --label_size LABEL_SIZE
                        Controls annotation label size. Must be an integer. Defaults to 8.
  --interval INTERVAL   Axis tick interval. Must be an integer. Must be an integer. Defaults to 5000.
  --truncate TRUNCATE   Number of characters to include in annoation labels before truncation with ellipsis. 
                        Must be an integer. Defaults to 20.
  --dpi DPI             Resultion (dots per inch). Must be an integer. Defaults to 600.
  --annotations ANNOTATIONS
                        Controls the proporition of annotations labelled. Must be a number between 0 and 1 inclusive. 
                        0 = no annotations, 0.5 = half of the annotations, 1 = all annotations. 
                        Defaults to 1. Chosen in order of CDS size.
  -t PLOT_TITLE, --plot_title PLOT_TITLE
                        Plot name.
  --label_ids LABEL_IDS
                        Text file with list of CDS IDs (from gff file) that are guaranteed to be labelled.

```

--------------------------------

### Run MMseqs2 Only Protein Annotation

Source: https://pharokka.readthedocs.io/en/latest/proteins

Use this command to run only MMseqs2 searches on PHROGs, CARD, and VFDB, excluding PyHMMER.

```bash
pharokka_proteins.py -i <input protein FASTA file> -d <database directory> -o <output folder> -t <threads> --mmseqs2_only
```

--------------------------------

### Pharokka Proteins Script Usage

Source: https://pharokka.readthedocs.io/en/latest/proteins

This shows the full usage of the pharokka_proteins.py script, including all available options and their descriptions.

```bash
usage: pharokka_proteins.py [-h] [-i INFILE] [-o OUTDIR] [-d DATABASE] [-t THREADS] [-f] [-p PREFIX] [-e EVALUE] [--hmm_only] [--mmseqs2_only] [-V] [--citation]

pharokka_proteins.py: fast phage protein annotation script

options:
  -h, --help            show this help message and exit
  -i INFILE, --infile INFILE
                        Input proteins file in amino acid fasta format.
  -o OUTDIR, --outdir OUTDIR
                        Directory to write the output to.
  -d DATABASE, --database DATABASE
                        Database directory. If the databases have been installed in the default directory, this is not required. Otherwise specify the path.
  -t THREADS, --threads THREADS
                        Number of threads. Defaults to 1.
  -f, --force           Overwrites the output directory.
  -p PREFIX, --prefix PREFIX
                        Prefix for output files. This is not required.
  -e EVALUE, --evalue EVALUE
                        E-value threshold for MMseqs2 database PHROGs, VFDB and CARD and pyhmmer PHROGs database search. Defaults to 1E-05.
  --hmm_only            Runs pyhmmer (HMMs) with PHROGs only, not MMseqs2 with PHROGs, CARD or VFDB.
  --mmseqs2_only        Runs MMseqs2 with PHROGs, CARD and VFDB only (same as Pharokka v1.3.2 and prior).
  -V, --version         Print pharokka Version
  --citation            Print pharokka Citation
```

--------------------------------

### Controlling Annotation Display Proportion

Source: https://pharokka.readthedocs.io/en/latest/plotting

Limits the number of CDS annotations displayed on the plot by specifying a proportion. Use 0.5 for half, 0 for none.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory --annotations 0.5
```

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory --annotations 0
```

--------------------------------

### Run Pharokka with alternative genetic codes

Source: https://pharokka.readthedocs.io/en/latest/run

Use this command to specify an alternative genetic code when running Pharokka with Prodigal as the gene predictor. The `-c` flag accepts a genetic code number.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  -g prodigal -c 4
```

--------------------------------

### Run Pharokka with MMseqs2 only

Source: https://pharokka.readthedocs.io/en/latest/run

Use this command to disable PyHMMER and only run MMseqs2 for analysis. This is useful for faster processing when PyHMMER is not required.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --mmseqs2_only
```

--------------------------------

### Run HMMs Only Protein Annotation

Source: https://pharokka.readthedocs.io/en/latest/proteins

Use this command to run only PyHMMER on the PHROGs database, excluding MMseqs2 searches.

```bash
pharokka_proteins.py -i <input protein FASTA file> -d <database directory> -o <output folder> -t <threads> --hmm_only
```

--------------------------------

### Meta Mode for Multiple Contigs

Source: https://pharokka.readthedocs.io/en/latest/run

When annotating more than one contig, enable meta mode with the `-m` flag. This optimizes thread usage for tRNAscan-SE 2, potentially speeding up the process.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  -m  -g prodigal-gv
```

--------------------------------

### Setting Plot Resolution (DPI)

Source: https://pharokka.readthedocs.io/en/latest/plotting

Specifies the resolution of the output PNG plot in dots per inch (DPI) using the `-dpi` option.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory -dpi 600
```

--------------------------------

### Skip Extra Annotations

Source: https://pharokka.readthedocs.io/en/latest/run

As of v1.5.0, you can skip tRNAscan-SE 2, MinCED, and Aragorn by using the `--skip_extra_annotations` flag.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --skip_extra_annotations
```

--------------------------------

### Specify custom mash distance threshold

Source: https://pharokka.readthedocs.io/en/latest/run

Set a custom mash distance threshold for comparison against the INPHARED database. This allows fine-tuning of similarity matching.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --mash_distance 0.05
```

--------------------------------

### Manually reorient phage using terminase coordinates (negative strand)

Source: https://pharokka.readthedocs.io/en/latest/run

Manually reorient the phage sequence to begin at a specific coordinate on the negative strand, typically the end coordinate of the large terminase subunit. Requires the input to be a single contig.

```bash
pharokka.py \
  -i <fasta file> \
  -o <output folder> \
  -d <path/to/database_dir> \
  -t <threads> \
  --terminase \
  --terminase_start 82226 \
  --terminase_strand neg
```

--------------------------------

### Specifying Locus Tags for Labeling

Source: https://pharokka.readthedocs.io/en/latest/plotting

Labels only specific features by providing a text file containing locus tags via the `--label_ids` option.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory --label_ids labels.txt
```

--------------------------------

### Use Prodigal-gv Gene Predictions

Source: https://pharokka.readthedocs.io/en/latest/run

For metagenomic datasets, it's recommended to use Prodigal-gv (pyrodigal-gv) for gene predictions, especially if phages might have alternate genetic codes. Use `-g prodigal-gv`.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  -g prodigal-gv
```

--------------------------------

### Skip Mash for INPHARED

Source: https://pharokka.readthedocs.io/en/latest/run

As of v1.5.0, you can skip the mash run for finding the closest contig match in INPHARED by using the `--skip_mash` flag.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  --skip_mash
```

--------------------------------

### Specify E-value Threshold

Source: https://pharokka.readthedocs.io/en/latest/run

Set a custom E-value threshold for CDS functional assignment using MMseqs2 with the `-e` flag. The default is 1E-5.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  -e <E-value>
```

--------------------------------

### Labeling Hypothetical Genes

Source: https://pharokka.readthedocs.io/en/latest/plotting

Includes labels for hypothetical or unknown genes on the plot, which are excluded by default.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory --label_hypotheticals
```

--------------------------------

### Customizing Axis Interval Size

Source: https://pharokka.readthedocs.io/en/latest/plotting

Sets the interval size for the plot's axes in base pairs using the `--interval` option.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory -t '${Escherichia}$ phage lambda' --title_size 30 --interval 10000
```

--------------------------------

### Adding a Custom Title to the Plot

Source: https://pharokka.readthedocs.io/en/latest/plotting

Assigns a custom title to the circular genome plot. Enclose the title in single quotes.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory -t 'phage lambda'
```

--------------------------------

### Adding Italicized Title Text

Source: https://pharokka.readthedocs.io/en/latest/plotting

Formats specific parts of the title with italics using `${ }$` delimiters, useful for scientific names.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory -t '${Escherichia}$ phage lambda'
```

--------------------------------

### Specify Custom Locus Tag

Source: https://pharokka.readthedocs.io/en/latest/run

To set a custom locus tag in the GFF/GenBank file, use the `-l` flag.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/databse_dir> -t <threads> -p <prefix>  -f -l <locus_tag>
```

--------------------------------

### Increasing CDS Label Font Size

Source: https://pharokka.readthedocs.io/en/latest/plotting

Adjusts the font size for CDS labels on the plot using the `--label_size` option.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory --label_size 10
```

--------------------------------

### Use Prodigal Gene Predictions

Source: https://pharokka.readthedocs.io/en/latest/run

To use Prodigal (pyrodigal) for gene predictions instead of PHANOTATE, specify `-g prodigal`.

```bash
pharokka.py -i <fasta file> -o <output folder> -d <path/to/database_dir> -t <threads>  -g prodigal
```

--------------------------------

### Increasing Title Font Size

Source: https://pharokka.readthedocs.io/en/latest/plotting

Adjusts the font size of the plot title using the `--title_size` option. The value specifies the point size.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory -t '${Escherichia}$ phage lambda' --title_size 30
```

--------------------------------

### Truncating Axis Label Length

Source: https://pharokka.readthedocs.io/en/latest/plotting

Limits the maximum length of axis labels to prevent overcrowding. Specify the desired character count.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory --truncate 15
```

--------------------------------

### Removing Labels for Other Features

Source: https://pharokka.readthedocs.io/en/latest/plotting

Disables labels for tRNAs, tmRNAs, and CRISPRs on the plot using the `--remove_other_features_labels` flag.

```bash
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory --remove_other_features_labels
```
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