PhiSiGns
~~~~~~~~
DESCRIPTION
-----------
A computational tool for the identification of phage signature genes and PCR primer design
PURPOSE
-------
PhiSiGns is intended for the phage biologists who are interested
1) in finding shared conserved genes in phages for evolution-based studies and/or
2) in designing primers on shared genes that can be used to discover unknown phages in the environment
AVAILABILTY
-----------
PhiSiGns is available at http://www.phantome.org/phisigns/ and http://phisigns.sourceforge.net/
CURRENT VERSION
---------------
PhiSiGns version 1.0
COPYRIGHT
---------
Copyright © 2011-2012, Bhakti Dwivedi
BUG REPORTS
-----------
Please report any bug(s) to me <bhaktihd_at_gmail_dot_com>
STANDALONE Vs. WEB-BASED VERSION
--------------------------------
PhiSiGns is a standalone and web-based application. Both versions provide the same features and results, but differ in the following:
1) The standalone version does not come with a phage genome database or precalculated BLASTP outputs. The user inputs the phage genomes to be compared for signature gene identification and primer design in an input txt file. The phage genomes are input as NCBI refseq numbers (see Standalone version - running mode). The GenBank files for the user-selected set of phage genomes are then obtained from NCBI and the gene annotations for the protein coding regions are imported accordingly. In contrast, the PhiSiGns web-based version consists of a database of 636 phages and 33 archaeal viruses (derived from the phage database on the PhAnToMe website, Feb 2011 [http://www.phantome.org/Downloads]), and pre-calculated BLASTP outputs for all these genomes (computed using PhiSiGns at an E-value cut-off of 10). The gene annotations are imported from the SEED [http://www.theseed.org/wiki/Home_of_the_SEED] and the proteins that lack annotation are extracted from GenBank. Therefore, with the standalone version users have the ability to compare phage genomes that are not part of the web-based PhiSiGns phage database.
2) The standalone version does not provide the option to select/deselect genes within the selected signature gene group for alignment and primer design. However, users can upload their own alignment containing only the sequences of interest.
STANDALONE VERSION - PREREQUISITES
----------------------------------
Standalone version is a perl script (phisigns.pl) and requires the following softwares and modules to run:
Resources
Direct internet connection
Software
BLASTALL (Blast standalone package)
CLUSTALW 1.8 (Multiple sequence alignment tool)
BioPerl modules
Bio::Seq
Bio::SeqIO
Bio::SeqFeature::Generic
Bio::DB::GenBank
Bio::DB::Fasta
Bio::Index::Fasta
Bio::Tools::Run::StandAloneBlast
Bio::Tools::Run::Alignment::Clustalw
Bio::Align::Utilities qw(aa_to_dna_aln)
Bio::AlignIO;
Perl modules
Getopt::Long
File::Basename
List::Util qw(max)
Files
input.txt
Also, you must have administrative rights to create, delete, read, or write files in the PhiSiGns working directory whereever located on your computer.
STANDALONE VERSION - SETTING VARIABLES
--------------------------------------
Following variables must be set before running phisigns.pl:
$BLASTALL (BLASTALL executable directory path)
Example: $BLASTALL = '/home/user/blast/bin/blastall'
$FORMATDB (FORMATDB executable directory path)
Example: $FORMATDB = '/home/user/blast/bin/formatdb'
$ENV{CLUSTALDIR} (CLUSTALW directory path)
Example: $ENV{CLUSTALDIR} = '/home/user/clustalw1.8/'
STANDALONE VERSION - COMMAND OPTIONS
------------------------------------
Option Description Default Argument
___________________________________________________________
-d PhiSiGns working directory path - string
-m run mode 0 int
-e BLAST E-value cut-off 10 float
-c BLAST coverage cut-off 10 float
-i input file - string
-g selected SiG_# for primer design - string
-a user uploaded alignment file - string
-aln_only Display default CLUSTALW alignment - none
-id id of current job - string
STANDALONE VERSION - RUNNING MODE
---------------------------------
There are two modes to run PhiSiGns (phisigns.pl):
MODE 0
------
Identifies the signature genes amongst the user-selected phage genomes as listed in the input file ('-i') saved under the PhiSiGns working directory. The input file must include the NCBI refseq number for the phage genomes to be compared for signature gene identification and primer design. Type the following command line to execute phisigns.pl in Mode = 0:
Example: % phisigns.pl -d /home/user/phisigns/ -m 0 -e 0.1 -c 25 -i input.txt -id test
BLAST E-value cut-off ('-e') and coverage ('-c') cut-off can be set by the user, if not specified a default value of 10 and 10 is used, respectively. Once the above command is executed and finished running, data and result files are generated with respect to the job id specified ('-id') in the command line under the PhiSiGns working directory.
Output files:
data/.blastoutput BLASTALL results for the selected phage genomes
data/.cdstbl.ffd Phage CDS table
data/.cds_nucleotide.fa CDS Nucleotide sequences
output/.sig_gps.txt short list of identified signature genes (SiG's)
output/.sig_gps_detail.txt detailed list of SiG's
output/.log.txt phisigns log file
MODE 1
------
Design PCR primer pairs for a selected signature gene (SiG_#) from the list of identified SiG's ('.sig_gps.txt' or .sig_gps_detail.txt' file) generated in Mode = 0. The primers are designed taking into account the minimum and maximum values specified for the primer parameters as in the pparams.txt file. Users can modify the values of these parameters to their preference.
Primer Parameters options
Flag Description Default Range
_________________________________________________________________________
minl min primer length 16 10-28
maxl max primer length 28 10-28
mingc min GC content 30 20-80
maxgc max GC content 80 30-80
minbtm min Basic Melting Temperature 30 30-80
maxbtm max Basic Melting Temperature 80 30-80
minstm min Salt Adjusted Temperature 30 30-80
maxstm max Salt Adjusted Temperature 80 30-80
minnnh min Nearest Neighbor Temperature 30 30-80
maxnnh max Nearest Neighbor Temperature 80 30-80
mindg min delta G for a primer -20 -30-0
minpl min product length 400 100-2500
maxpl max product length 2000 100-2500
dgn primer degeneracy 1000 1-1000
gcclamp 3' GC clamp y y or n
max3stb maximum 3' stability y y or n
compl Complementarity y y or n
Type the following command line to execute phisigns.pl in Mode = 1 to only display program generated alignment for a selected signature gene:
Example: % phisigns.pl -d /home/user/phisigns/ -m 1 -g SiG_5 -aln_only -id test
Type the following command line to execute phisigns.pl in Mode = 1 with the program generated alignment:
Example: % phisigns.pl -d /home/user/phisigns/ -m 1 -g SiG_5 -id test
Users can also upload their own nucleotide sequence alignment (in CLUSTALW format only with '.aln' file extension) for the selected signature gene group to design PCR primer pairs. The user uploaded alignment file should be under the phisigns working directory.
Type the following command line to execute phisigns.pl in Mode = 1 with a user-generated alignment:
Example: % phisigns.pl -d /home/user/phisigns/ -m 1 -a test.aln -id test
Once the above command is executed and finished running, results files for the user uploaded alignment are generated with respect to the job id specified ('-id') in the command line under the phisigns working directory.
Output files:
output/.seqs sequences in FASTA format for the user-selected SiG_#
output/.aln nucleotide sequence CLUSTALW alignment for the selected SiG_#
output/.pairs.txt short list of potential primer pairs found for the selected SiG_#
output/.pairs_detail.txt detailed list of the potential primer pairs found for the selected SiG_#
output/.log.txt phisigns log file
Note: PhiSiGns (phisigns.pl) in Mode = 1 will execute only after PhiSiGns is executed in Mode = 0. Unless the selected set of phage genomes is different, Mode = 0 need not be executed more than once. To design PCR primer pairs with a user-uploaded alignment Mode = 1 can be executed without running Mode = 0 first.
WEB VERSION - RUNNING
----------------------
A. Identify signature genes
1. Select and add phage genomes of interest from the list of available phage genomes
2. Select BLAST E-value and coverage cut-off for similarity searches
3. Click on "Get Signature Genes"
4. View the list on the webpage or download the table on a local machine
B. Design PCR primer pairs
1. Select a signature gene from the list of identified signature genes amongst the phage genomes of interest
2. Select minimum and maximum primer parameter values [optional]
3. Select/deselect genes within the selected signature gene group for alignment and primer design [optional]
4. View program generated CLUSTALW alignment for selected genes [optional]
5. Upload your own nucleotide sequence alignment for the selected signature gene [optional]
6. Click on "Design Primers for Selected SiG Genes"
7. View the results on the webpage or download them on a local machine
For more details on web version, please see http://phisigns.sourceforge.net/
WEB VERSION - DEPENDENCIES
--------------------------
The perl script require these Perl modules:
CGI qw(:standard :html3)
CGI::Carp qw(fatalsToBrowser)
Fcntl qw(:DEFAULT :flock)
