Extra special thanks to Ghais Houtak for making Pharokka's logo.
pharokka
is a rapid standardised annotation tool for bacteriophage genomes and metagenomes.
If you are looking for rapid standardised annotation of bacterial genomes, please use Bakta. Prokka, which inspired the creation and naming of pharokka
, is another good option, but Bakta is Prokka's worthy successor.
If you like pharokka
, you will probably love phold. phold
uses structural homology to improve phage annotation. Benchmarking is ongoing but phold
strongly outperforms pharokka
in terms of annotation, particularly for less characterised phages such as those from metagenomic datasets.
pharokka
still has features phold
lacks for now (identifying tRNA, tmRNA, CRISPR repeats, and INPHARED taxonomy search), so it is recommended to run phold
after running pharokka
.
phold
takes the Genbank output of Pharokka as input. Therefore, if you have already annotated your phage(s) with Pharokka, you can easily update the annotation with more functional predictions with phold.
If you don't want to install pharokka
or phold
locally, you can run pharokka
and phold
(and phynteny
), or only pharokka
, without any code using the Google Colab notebook.
phynteny
uses a long-short term memory model trained on phage synteny (the conserved gene order across phages) to assign hypothetical phage proteins to a PHROG category - it might help you add extra PHROG category annotations to hypothetical genes remaining after you run phold
. pharokka
and/or phold
if your phage(s) are too big - just don't run the Phynteny step!The easiest way to install pharokka
is via conda:
conda install -c bioconda pharokka
Followed by database download and installation:
install_databases.py -o <path/to/databse_dir>
And finally annotation:
pharokka.py -i <phage fasta file> -o <output directory> -d <path/to/database_dir> -t <threads>
As of pharokka
v1.4.0, if you want extremely rapid PHROG annotations, use --fast
:
pharokka.py -i <phage fasta file> -o <output directory> -d <path/to/database_dir> -t <threads> --fast
Check out the full documentation at https://pharokka.readthedocs.io.
pharokka
has been published in Bioinformatics:
George Bouras, Roshan Nepal, Ghais Houtak, Alkis James Psaltis, Peter-John Wormald, Sarah Vreugde, Pharokka: a fast scalable bacteriophage annotation tool, Bioinformatics, Volume 39, Issue 1, January 2023, btac776, https://doi.org/10.1093/bioinformatics/btac776.
If you use pharokka
, please see the full Citation section for a list of all programs pharokka uses, in order to fully recognise the creators of these tools for their work.
Thanks to some amazing assistance from Paul Zierep, you can run pharokka
using the Galaxy Europe webserver. There is no plotting functionality at the moment.
So if you can't get pharokka
to install on your machine for whatever reason or want a GUI to annotate your phage(s), please give it a go there.
pharokka
uses PHANOTATE, the only gene prediction program tailored to bacteriophages, as the default program for gene prediction. Prodigal implemented with pyrodigal and Prodigal-gv implemented with pyrodigal-gv are also available as alternatives. Following this, functional annotations are assigned by matching each predicted coding sequence (CDS) to the PHROGs, CARD and VFDB databases using MMseqs2. As of v1.4.0, pharokka
will also match each CDS to the PHROGs database using more sensitive Hidden Markov Models using PyHMMER. Pharokka's main output is a GFF file suitable for using in downstream pangenomic pipelines like Roary. pharokka
also generates a cds_functions.tsv
file, which includes counts of CDSs, tRNAs, tmRNAs, CRISPRs and functions assigned to CDSs according to the PHROGs database. See the full usage and check out the full documentation for more details.
You can run pharokka_multiplotter.py
to plot as many phage(s) as you want.
It requires the pharokka
output Genbank file (here, pharokka.gbk
). It will save plots for each contig in the output directory (here pharokka_plots_output_directory
).
e.g.
pharokka_multiplotter.py -g pharokka.gbk -o pharokka_plots_output_directory
pharokka_proteins.py
crashing if it found VFDB hits, #303 errors in the .tbl
format, #316 errors with types and where custom HMM dbs had identical scored hits, #317 types and #320 deprecated GC function)--mash_distance
and --minced_args
as parameters (#299 thanks @iferres).pyrodigal-gv
implementing prodigal-gv
as a gene predictor for alternate genetic codes (pyrodigal-gv and prodigal-gv). This can be specified with -g prodigal-gv
and is recommended for metagenomic input datasets. Thanks to @althonos and @apcamargo for making this possible, and to @asierFernandezP for raising this as an issue in the first place here.-g prodigal
and -g prodigal-gv
should be much faster thanks to multithread support added by the inimitable @althonos._length_gc_cds_density.tsv
and _cds_final_merged_output.tsv
files now contain the translation table/genetic code for each contig.--skip_mash
flag added to skip finding the closest match for each contig in INPHARED using mash.--skip_extra_annotations
flag added to skip running tRNA-scanSE, MinCED and Aragorn in case you only want CDS predictions and functional annotations.pharokka
v1.4.0 is a large update implementing:
pharokka
will now run searches using both MMseqs2 (PHROGs, CARD and VFDB) and HMMs (PHROGs). MMseqs2 was kept for PHROGs as it provides more information than the HMM results (e.g. sequence alignment identities & top hit PHROG protein) if it finds a hit. --fast
or --hmm_only
which only runs PyHMMER on PHROGs. It will not run MMseqs2 at all on PHROGs, CARD or VFDB. For phage isolates, this will be much faster than v1.3.2, but you will not get CARD or VFDB annotations. For metagenomes, this will be (much) slower though!pharokka
v1.4.0 run somewhat faster than v1.3.2, even if PyHMMER is not used and --mmseqs2_only
is specified.pharokka
v1.4.0 databases. The VFDB database is now clustered at 50% sequence identity (which speeds up runtime).--mmseqs2_only
which will essentially run pharokka
v1.3.2 and is default in meta mode -m
or --meta
. pharokka_proteins.py
, which takes an input file of amino acid proteins in FASTA format and runs MMseqs2 (PHROGs, CARD, VFDB) and PyHMMER (PHROGs). See the proteins documentation for more details.--custom_hmm
, which allows for custom HMM profile databases to be used with pharokka
.create_custom_hmm.py
which facilitates the creation of a HMM profile database from multiple sequence alignments. See the documentation for more details about how to create a compatible HMM profile database.--dnaapler
, which automatically detects and reorients your phage to start with the large terminase subunit. For more information, see dnaapler.--genbank
, which allows for genbank format input with -i
. This will take all (custom) CDS calls in genbank file and PHANOTATE/pyrodigal will not be run. So if you have done manual gene curation, this option is recommended.-c
, which should now work with -g prodigal
(thanks Alistair Legione for the fixes).pharokka
v1.3.0 implements pharokka_plotter.py
, which creates a simple circular genome plot using the amazing pyCirclize package with output in PNG format. All CDS are coloured according to their PHROG functional group.
It is reasonably customisable and is designed for single input phage contigs. If an input FASTA with multiple contigs is entered, it will only plot the first contig.
It requires the input FASTA, pharokka
output directory, and the -p
or --prefix
value used with pharokka
if specified.
You can run pharokka_plotter.py
in the following form
pharokka_plotter.py -i input.fasta -n pharokka_plot -o pharokka_output_directory
This will create pharokka_plot.png
as an output file plot of your phage.
An example plot is included below made with the following command (assuming Pharokka has been run with SAOMS1_pharokka_output_directory
as the output directory).
pharokka_plotter.py -i test_data/SAOMS1.fasta -n SAOMS1_plot -o SAOMS1_pharokka_output_directory --interval 8000 --annotations 0.5 --plot_title '${Staphylococcus}$ Phage SAOMS1'
SAOMS1 phage (GenBank: MW460250.1) was isolated and sequenced by: Yerushalmy, O., Alkalay-Oren, S., Coppenhagen-Glazer, S. and Hazan, R. from the Institute of Dental Sciences and School of Dental Medicine, Hebrew University, Israel.
Please see plotting for details on all plotting parameter options.
The easiest way to install pharokka
is via conda. For inexperienced command line users, this method is highly recommended.
conda install -c bioconda pharokka
This will install all the dependencies along with pharokka
. The dependencies are listed in environment.yml.
If conda is taking a long time to solve the environment, try using mamba:
conda install mamba
mamba install -c bioconda pharokka
As of v1.4.0, you can also install the python components of pharokka
with pip.
pip install pharokka
You will still need to install the non-python dependencies manually.
If you have Docker/Singularity/Apptainer installed, you can use the biocontainers container (yes, every bioconda package has one!)
You might find this useful if you have trouble with conda environments.
For example to install pharokka v1.7.3
with Singularity:
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 $IMAGE_DIR docker://quay.io/biocontainers/pharokka:1.7.3--pyhdfd78af_0
singularity exec <.sif file>
e.g.:containerImage="$IMAGE_DIR/pharokka_1.7.3--pyhdfd78af_0.sif"
singularity exec $containerImage pharokka.py -h
Alternatively, the development version of pharokka
(which may include new, untested features) can be installed manually via github.
git clone https://github.com/gbouras13/pharokka.git
cd pharokka
pip install -e .
pharokka.py --help
The dependencies found in environment.yml will then need to be installed manually.
For example using conda to install the required dependencies:
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
To install the pharokka
database to the default directory:
install_databases.py -d
If you would like to specify a different database directory (recommended), that can be achieved as follows:
install_databases.py -o <path/to/databse_dir>
If this does not work, you an alternatively download the databases from Zenodo at https://zenodo.org/record/8276347/files/pharokka_v1.4.0_databases.tar.gz and untar the directory in a location of your choice.
If you prefer to use the command line:
wget "https://zenodo.org/record/8276347/files/pharokka_v1.4.0_databases.tar.gz"
tar -xzf pharokka_v1.4.0_databases.tar.gz
which will create a directory called "pharokka_v1.4.0_databases" containing the databases.
If you are new to using the command-line, please install conda using the following instructions.
curl -O https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
For Mac (Intel, will also work with M1):
curl -O https://repo.anaconda.com/miniconda/Miniconda3-latest-MacOSX-x86_64.sh
sh Miniconda3-latest-Linux-x86_64.sh
conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge
conda install mamba
pharokka
into a fresh environment. For example to create an environment called pharokkaENV with pharokka
installed:mamba create -n pharokkaENV pharokka
conda activate pharokkaENV
install_databases.py -h
pharokka.py -h
Once the databases have finished downloading, to run pharokka
:
pharokka.py -i <fasta file> -o <output directory> -t <threads>
To specify a different database directory (recommended):
pharokka.py -i <fasta file> -o <output directory> -d <path/to/database_dir> -t <threads> -p <prefix>
For a full explanation of all arguments, please see usage.
pharokka defaults to 1 thread.
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.
-V, --version Print pharokka Version
--citation Print pharokka Citation
A brief description of what is new in each update of pharokka
can be found in the HISTORY.md file.
pharokka
has been tested on Linux and MacOS (M1 and Intel).
On a standard 16GB RAM laptop specifying 8 threads, pharokka
should take between 3-10 minutes to run for a single phage, depending on the genome size.
In --fast
mode, it should take 45-75 seconds.
pharokka v1.5.0
was run on the 673 crAss phage dataset to showcase the improved CDS prediction of -g prodigal-gv
for metagenomic datasets where some phages likely have alternative genetic codes (i.e. not 11).
All benchmarking was conducted on a Intel® Core™ i7-10700K CPU @ 3.80GHz on a machine running Ubuntu 20.04.6 LTS with 8 threads (-t 8
). pyrodigal-gv v0.1.0
and pyrodigal v3.0.0
were used respectively.
673 crAss-like genomes | pharokka v1.5.0 -g prodigal-gv |
pharokka v1.5.0 -g prodigal |
---|---|---|
Total CDS | 81730 | 91999 |
Annotated Function CDS | 20344 | 17458 |
Unknown Function CDS | 61386 | 74541 |
Contigs with genetic code 15 | 229 | NA |
Contigs with genetic code 4 | 38 | NA |
Contigs with genetic code 11 | 406 | 673 |
Fewer (larger) CDS were predicted more accurately, leading to an increase in the number of coding sequences with annotated functions. Approximately 40% of contigs in this dataset were predicted to use non-standard genetic codes according to pyrodigal-gv
.
pharokka
v1.4.0 has also been run on phage SAOMS1 and also the same 673 crAss phage dataset to showcase:
--fast
is slower for metagenomes.
--fast
or --meta_hmm
for metagenomes given how much more sensitive HMM search is.--fast
for phage isolates - with the proviso that no virulence factors or AMR genes will be detected. --mmseqs2_only
.All benchmarking was conducted on a Intel® Core™ i7-10700K CPU @ 3.80GHz on a machine running Ubuntu 20.04.6 LTS with 16 threads (-t 16
).
SAOMS1 was run with Phanotate
Phage SAOMS1 | pharokka v1.4.0 --fast |
pharokka v1.4.0 |
pharokka v1.3.2 |
---|---|---|---|
Time (min) | 0.70 | 3.73 | 5.08 |
CDS | 246 | 246 | 246 |
Annotated Function CDS | 93 | 93 | 92 |
Unknown Function CDS | 153 | 153 | 154 |
The 673 crAss-like genomes were run with -m
(defaults to --mmseqs2_only
in v 1.4.0) and with -g prodigal
(pyrodigal v2.1.0).
673 crAss-like genomes | pharokka v1.4.0 --fast |
pharokka v1.4.0 --mmseqs2_only |
pharokka v1.3.2 |
---|---|---|---|
Time (min) | 35.62 | 11.05 | 13.27 |
CDS | 91999 | 91999 | 91999 |
Annotated Function CDS | 16713 | 9150 | 9150 |
Unknown Function CDS | 75286 | 82849 | 82849 |
pharokka
(v1.1.0) has been benchmarked on an Intel Xeon CPU E5-4610 v2 @ 2.30 specifying 16 threads. Below is benchamarking comparing pharokka
run with PHANOTATE and Prodigal against Prokka v1.14.6 run with PHROGs HMM profiles, as modified by Andrew Millard (https://millardlab.org/2021/11/21/phage-annotation-with-phrogs/).
Benchmarking was conducted on Enterbacteria Phage Lambda (Genbank accession J02459) Staphylococcus Phage SAOMS1 (Genbank Accession MW460250) and 673 crAss-like phage genomes in one multiFASTA input taken from Yutin, N., Benler, S., Shmakov, S.A. et al. Analysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAss-like phages with unique genomic features. Nat Commun 12, 1044 (2021) https://doi.org/10.1038/s41467-021-21350-w.
For the crAss-like phage genomes, pharokka
meta mode -m
was enabled.
Phage Lambda | pharokka PHANOTATE |
pharokka Prodigal |
Prokka with PHROGs |
---|---|---|---|
Time (min) | 4.19 | 3.88 | 0.27 |
CDS | 88 | 61 | 62 |
Coding Density (%) | 94.55 | 83.69 | 84.96 |
Annotated Function CDS | 43 | 37 | 45 |
Unknown Function CDS | 45 | 24 | 17 |
Phage SAOMS1 | pharokka PHANOTATE |
pharokka Prodigal |
Prokka with PHROGs |
---|---|---|---|
Time (min) | 4.26 | 3.89 | 0.93 |
CDS | 246 | 212 | 212 |
Coding Density (%) | 92.27 | 89.69 | 89.31 |
Annotated Function CDS | 92 | 93 | 92 |
Unknown Function CDS | 154 | 119 | 120 |
673 crAss-like genomes from Yutin et al., 2021 | pharokka PHANOTATE Meta Mode |
pharokka Prodigal Meta Mode |
Prokka with PHROGs |
---|---|---|---|
Time (min) | 106.55 | 11.88 | 252.33 |
Time Gene Prediction (min) | 96.21 | 3.4 | 5.12 |
Time tRNA Prediction (min) | 1.25 | 1.08 | 0.3 |
Time Database Searches (min) | 6.75 | 5.58 | 238.77 |
CDS | 138628 | 90497 | 89802 |
Contig Min Coding Density (%) | 66.01 | 46.18 | 46.13 |
Contig Max Coding Density (%) | 98.86 | 97.85 | 97.07 |
Annotated Function CDS | 9341 | 9228 | 14461 |
Unknown Function CDS | 129287 | 81269 | 75341 |
pharokka
scales well for large metavirome datasets due to the speed of MMseqs2. In fact, as the size of the input file increases, the extra time taken is required for running gene prediction (particularly PHANOTATE) and tRNA-scan SE2 - the time taken to conduct MMseqs2 searches remain small due to its many vs many approach.
If you require fast annotations of extremely large datasets (i.e. thousands of input contigs), running pharokka
with Prodigal (-g prodigal
) is recommended.
If you come across bugs with pharokka
, or would like to make any suggestions to improve the program, please open an issue or email george.bouras@adelaide.edu.au.
George Bouras, Roshan Nepal, Ghais Houtak, Alkis James Psaltis, Peter-John Wormald, Sarah Vreugde, Pharokka: a fast scalable bacteriophage annotation tool, Bioinformatics, Volume 39, Issue 1, January 2023, btac776, https://doi.org/10.1093/bioinformatics/btac776
If you use pharokka
, I would recommend a citation in your manuscript along the lines of:
With the following full citations for the constituent tools below where relevant: