#What is PhiSpy?

PhiSpy identifies prophages in Bacterial (and probably Archaeal) genomes. Given an annotated genome it will use several approaches to identify the most likely prophage regions.

Initial versions of PhiSpy were written by

Sajia Akhter (sajia@stanford.edu) Edwards Bioinformatics Lab

Improvements, bug fixes, and other changes were made by

Katelyn McNair Edwards Bioinformatics Lab and Przemyslaw Decewicz University of Warsaw

Software Requirements

PhiSpy requires following programs to be installed in the system. Most of these are likely already on your system.

1. Python - version 3.4 or later
2. Biopython - version 1.58 or later
3. gcc - GNU project C and C++ compiler - version 4.4.1 or later
4. The Python.h header file. This is included in python3-dev that is available on most systems.

INSTALLATION

For a brand new Ubuntu installation (e.g. on Google Cloud Platform you can install these dependencies with these commands:

sudo apt install -y build-essential python3-dev python3-pip
python3 -m pip install --user PhiSpy

This will install PhiSpy.py in ~/.local/bin which should be in your $PATH but might not be (see this detailed discussion).

If you try PhiSpy.py -v and get an error like this:

$ PhiSpy.py -v
-bash: PhiSpy.py: command not found

Then you can either use the full path:

~/.local/bin/PhiSpy.py -v

or add that location to your $PATH:

echo "export PATH=\$HOME/.local/bin:\$PATH" >> ~/.bashrc
source ~/.bashrc
PhiSpy.py -v

Advanced Users

For advanced users, you can clone the git repository and use that (though pip is the recommended install method).

git clone https://github.com/linsalrob/PhiSpy.git
cd PhiSpy`
python3 setup.py install --user

If you have root and you want to install globally, you can change the setup command.

git clone https://github.com/linsalrob/PhiSpy.git
cd PhiSpy`
python3 setup.py install

For ease of use, you may wish to add the location of PhiSpy.py to your $PATH.

Testing PhiSpy.py

Download the Streptococcus pyogenes M1 genome

curl -Lo Streptococcus_pyogenes_M1_GAS.gb https://bit.ly/37qFArb
PhiSpy.py -o Streptococcus.phages Streptococcus_pyogenes_M1_GAS.gb

or to run it with the Streptococcus training set:

PhiSpy.py -o Streptococcus.phages -t data/trainSet_160490.61.txt Streptococcus_pyogenes_M1_GAS.gb

This uses the GenBank format file for Streptococcus pyogenes M1 GAS that we provide in the tests/ directory, and we use the training set for S. pyogenes M1 GAS that we have pre-calculated. This quickly identifies the four prophages in this genome, runs the repeat finder on all of them, and outputs the answers.

You will find the output files from this query in output_directory.

Running PhiSpy.py

The simplest command is:

PhiSpy.py -f genbank_file -o output_directory

where:

• genbank file: The input DNA sequence file in GenBank format.
• output directory: The output directory is the directory where the final output file will be created.

If you have new genome, we recommend annotating it using the RAST server or PROKKA.

After annotation, you can download the genome directory from the server.

Help

For the help menu use the -h option:

python PhiSpy.py -h

Output Files

There are 3 output files, located in output directory.

1. prophage.tbl: This file has two columns separated by tabs [id, location]. The id is in the format: pp_number, where number is a sequential number of the prophage (starting at 1). Location is be in the format: contig_start_stop that encompasses the prophage.

2. prophage_tbl.tsv: This is a tab seperated file. The file contains all the genes of the genome. The tenth colum represents the status of a gene. If this column is 1 then the gene is a phage like gene; otherwise it is a bacterial gene.

This file has 16 columns:(i) fig_no: the id of each gene; (ii) function: function of the gene; (iii) contig; (iv) start: start location of the gene; (v) stop: end location of the gene; (vi) position: a sequential number of the gene (starting at 1); (vii) rank: rank of each gene provided by random forest; (viii) my_status: status of each gene based on random forest; (ix) pp: classification of each gene based on their function; (x) Final_status: the status of each gene. For prophages, this column has the number of the prophage as listed in prophage.tbl above; If the column contains a 0 we believe that it is a bacterial gene. If we can detect the att sites, the additional columns will be: (xi) start of attL; (xii) end of attL; (xiii) start of attR; (xiv) end of attR; (xv) sequence of attL; (xvi) sequence of attR.

3. prophage_coordinates.tsv: This file has the prophage ID, contig, start, stop, and potential att sites identified for the phages.

Example Data

• Streptococcus pyogenes M1 GAS which has a single genome contig. The genome contains four prophages.

To analyze this data, you can use:

PhiSpy.py -o output_directory -t data/trainSet_160490.61.txt tests/Streptococcus_pyogenes_M1_GAS.gb

And you should get a prophage table that has this information (for example, take a look at output_directory/prophage.tbl).