BYU-Phamerator

This is not the official version of Phamerator.

To access the SEA-PHAGES online version of Phamerator, please visit http://phamerator.org.

Overview

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This is a fork being worked on by Brigham Young University Life Sciences and is NOT the official Phamerator repository.

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Phamerator is a set of computer programs that is useful for genomic analysis of bacteriophage genomes. In fact, these programs were specifically designed for this purpose.

Phamerator is released under a free software license. See LICENSE.txt for more details.

Documentation for this project can be found in the doc directory. A quick start guide is included in this README file.

This project was forked under GNU License v2 section 2 as above, BYU Life Sciences Information Technology.

Publications

Merrill and Ward et al. BMC Genomics, 2016 - https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-016-3018-2

Contact information

If you have a problem installing or using this GitHub repository, please open an issue here or contact the corresponding authors of Merrill and Ward et al. (2016). Do not contact the original creators of Phamerator, as they will not be able to help with this fork.

Requirements

For the server backend, you will need

• Python 2.7.5+
• Biopython 1.56 or newer (python-biopython)
• Pyro 3.14 (pyro) (note that version 4.x does not work as it is for Python 3.x)
• ClustalW (clustalw) - this package is located in non-free for Debian distributions
• ClustalOmega (clustalo) - also non-free, only required if using phamClientOmega.py
• BLAST (ftp://ftp.ncbi.nih.gov/blast/executables/release/LATEST)
• BLAST+ (ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/) (ncbi-blast+)
• Parallel Python (python-pp)
• MySQL for Python (python-mysqldb)
• curl
• MySQL Server (mysql-server)

In addition, for the GUI you will need

• Python GooCanvas Bindings (python-pygoocanvas)
• Python Webkit Bindings (python-webkit)
• Python Gnome configuration Bindings (python-gconf)
• GTK+ 3 or newer

apt-get install python-biopython pyro clustalw clustalo ncbi-blast+ python-pp python-mysqldb python-pygoocanvas python-webkit python-gconf

Phamerator Quick-Start Guide

These instructions are intended to get you up and running with Phamerator. For more complete documentation for each script, please see usage instructions, usually by running the script in question with no arguments. Before continuing with this guide, be sure to install all the required dependencies at the beginning of this document.

Additional information about this forked project can be found at https://phagehunters.byu.edu/Phamerator.

Downloading Phamerator

This Phamerator repository uses a program called Git. In order to download it, it is recommended that you first install Git, in order to facilitate quick updates of the Phamerator program. To install Git on a Debian-based Linux system (including Ubuntu), run the command:

sudo apt-get install git

After installing Git, you can download the latest version of this branch of Phamerator by running this command, which will download to the current working directory:

git clone https://github.com/byuphamerator/phamerator-dev.git

After downloading Phamerator with Git, in order to check for updates, simply change into the new phamerator directory that was downloaded and then run:

git pull

and Git will check for any updates that might have been made since you last downloaded files from the repository.

Setting Up the Database

Before Phamerator can be used a database back-end must be set up to accept new phage records. Phamerator uses MySQL. We will set up a new database, and then write the Phamerator schema to that new database. Finally, we will grant permissions to a new user on that database.

First, let’s create the database. During installation of mysql-server you should have created a root password for your database through a prompt in the installer. Run this command and enter that root password when prompted:

mysql -u root -p

If login is successful, you will be greeted with the MySQL prompt. At the prompt, run these commands to create a new database and grant access to a user for Phamerator:

mysql> CREATE DATABASE $database$;

mysql> GRANT SELECT, DROP, DELETE, INSERT, CREATE, LOCK TABLES, UPDATE on 
$database$.* to $username$ IDENTIFIED BY “$password$”;

mysql> FLUSH PRIVILEGES;

Replace $database$ with the name of your new database, $username$ with the name of your new user, and $password$ with the password for your new user. $database$ should be the same in both commands. If no errors result, we can exit the command prompt:

mysql> quit

To finish our database installation, we need to dump the database schema into our new database. The schema for Phamerator is included with the Phamerator files, in $phamerator-repo$/phamerator/sql/db_schema.sql, where $phamerator-repo$ is the name of the folder that you downloaded the Phamerator development snapshot repository. In order to install the schema, run this command on the command prompt:

mysql -u root -p $database$ < $phamerator-repo$/phamerator/sql/db_schema.sql

$database$ is the name of the database you created earlier, and $phamerator-repo$ is the name of the Phamerator repository directory, as before. Note the “<” character, pointing at your database name. You will be prompted for your root database password. If there is no error, your database installation is complete!

Entering Data Into the Phamerator Database

Phamerator data entry jobs typically take 5 steps:

1. Ensure compliance of GenBank files before database import
2. Add GenBank files to the database
3. Run genome alignments on sequences in the database using ClustalW and Blast
4. Build the Phams list and enter it into the database
5. Determine conserved domains using the NCBI Conserved Domain Database

Before any data entry can take place we must ensure that any GenBank files used are compliant with the format that Phamerator accepts. To do this, we will use the fix_dnaMaster_gb.py script, located in the phamerator/plugins directory. Collect your GenBank files into a directory and run the script on them like so:

./phamerator/plugins/fix_dnaMaster_gb.py /path/to/ImportSequences

where the first and only argument is a path to the directory containing your sequences you would like to import. The script will check the format of all GenBank files in that directory and write out a new version of each with a “.fixed” suffix.

Now we are ready to start importing GenBank files into the database. For this task we will use the phamerator_manage_db.py script, located in the phamerator directory. To import the GenBank files we checked in the previous step, run the command

./phamerator/phamerator_manage_db.py -u $username$ -p -s $database-server$ -d 
$database$ -i /path/to/ImportSequences

where $username$ is your database username, $database-server$ is the hostname of your database server, and $database$ is the name of your database.

Next it is time to run genome alignments on the newly added data. To do this, we will run the phamServer_InnoDB.py script, located in the phamerator directory. First, let’s run the server for ClustalW:

./phamerator/phamServer_InnoDB.py -u $username$ -p -s $database-server$ -n localhost -d $database$ -i 1 -l True -a clustalw

where $username$ is your database username, $database-server$ is the hostname of your database server, and $database$ is the name of your database. This will start an instance of the phamServer for ClustalW. In order to run any sequences, we will then have to connect to the server with a client. To do this, open another terminal, leaving the server running in your old one, and run:

./phamerator/phamClient.py -u $username$ -p -n localhost

If you would like to use the new Clustal Omega implementation, do

./phamerator/phamClientOmega.py -u $username$ -p -n localhost

where $username$ is your database username. This script will run alignments that the phamServer deals out.

In order to run blast jobs, repeat the previous server step, but with “blast” for the -a argument on the phamServer_InnoDB.py script. To run blast jobs, use the blastclient.py script, in the phamerator directory, like so:

./phamerator/blastclient.py -u $username$ -p -n localhost -a $path-to-blast$ -d $path-to-blast-data$

where $username$ is your database username, $path-to-blast$ is the path to your Blast binaries, and $path-to-blast-data$ is your Blast datapath.

Depending on the amount of data to be processed, ClustalW and Blast jobs can take a very long time, so remember to be very patient!

Next we will build the Pham list and import it into the database. To do this, we use the phamBuilder4.py script in the phamerator directory. Values for the -c and -b options will depend on your analysis. They define the cutoff points that make a protein acceptable to join the Pham being built. Some sample values are provided below. See the script usage for more information. Run the command like this:

./phamerator/phamBuilder4.py -u $username$ -p -s $database-server$ -d $database$ -c 0.325 -b 1e-50

where $username$ is your database username, $database-server$ is the hostname of your database server, and $database$ is the name of your database. This process also takes a long time, so now is a great time to take a break.

Finally, you have the option of determining conserved domains for the Phams in the database using the NCBI CDD database. For this to work, you will need to get a copy of the CDD database from NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). To run a search, use the cddSearch.py script, located in the phamerator/plugins directory. For usage, please run the script with no options.

Phamerator GUI

The Phamerator GUI is easy to use. If you already have a database set up by following the instructions above, all you have to do is start the GUI by running ./phamerator/Phamerator on the command line. Before the GUI starts you will be prompted for your database information. After a successful login, the Phamerator GUI will appear.

The first time that you run the Phamerator GUI, it will create a configuration file for itself in your home folder, at ~/.phamerator/phamerator.conf. This file contains sensible defaults, but they will need to be changed you have extra databases you would like to connect to (otherdatabases) or if you would like to change your Blast directory (blast_dir). Currently, the prompts before the GUI start override the settings in the configuration file for the default database and default server.

If you don’t have the required permissions on your database for the Phamerator GUI but you have the root password for your database, simply start Phamerator as above and enter your desired username and password. Phamerator will prompt you for your root password and attempt to add your username and password to the allowed list of users for the database.

One of Phamerator’s features is running local Blast jobs on entries in the database. In order to do that you will have to have a local copy of the Blast binaries on your machine. If Phamerator does not detect the required binaries on your machine on start it will download them for you into the directory specified by blast_dir in the configuration file.