FullSSR Files

FULSSR_v1.5
###################################################################
Traditional methods for isolation of microsatellites (SSRs) are often 
expensive and time consuming.
Nowadays is easier and cheaper to obtain genomic data from next 
generation sequencing. 
Then, at least two software need to be applied in order to obtain 
SSR primers of SSR flanking sequences for PCR assays. 
Here we propose new software that allows the user to work with several 
sequences for PCR primer design in one step.
###################################################################



Requirements
------------------

-Unix OS
-Perl
-Bioperl (from github, instructions in https://github.com/bioperl/bioperl-live/blob/master/README.md)
-Primer3 (download from https://github.com/primer3-org/primer3/releases)


Instalation
------------------

-Unzip FullSSR.tar.gz, from command prompt: 
	
	$ tar -zxvf FullSSR.tar.gz

-Get into the folder:
	
	$cd FullSSR

-Replace Primer3.pm file from /usr/share/perl5/Bio/Tools/Run/ with the one provided in FullSSR folder:
	 
	#The location of Bio/Tools/Run folder may vary between linux distribution, 
	#to check this use the commands below:
	
	$sudo updatedb
	$locate Primer3.pm	
	
	#copy the file FullSSR/Primer3.pm to /usr/share/perl5/Bio/Tools/Run/ (for this step you must have root permision):
	
	$sudo cp -r Primer3.pm /usr/share/perl5/Bio/Tools/Run/

-Open FullSSR perl script and specify the path where FullSSR was extracted:
	
	#Example
	require '/home/ubuntu/Desktop/FullSSR/parseprimer3.pm';
        require '/home/ubuntu/Desktop/FullSSR/readparameters.pm';
	
-Open config.txt and specify the path of primer3_core (this file is in primer3/src/ folder) and primer3_config/ folder:
	
	#Example
	>Primer3_PATH: /home/ubuntu/Desktop/primer3/src/primer3_core

	>Primer3_config_PATH: /home/ubuntu/Desktop/primer3/src/primer3_config/



Configure parameters for primer design
----------------
The parameters used for Primer3 are specified in config.txt file. 
These are the default parameters, you can modify them.
If you want more information about this parameters read 
the Primer3 manual (primer3/primer3_manual.htm).


How to execute
----------------

-Put the sequence file you want to analyze into the FullSSR folder (sequence must be in FASTA format)

-Open command prompt, and go to FullSSR folder:

	$cd FullSSR

-Execute perl script FullSSR.pl, you need to specify the sequence file name and the minimum motif repeats:
	
	#Execution example: filename sequence.fasta and 5 minimun repeats
	$perl FullSSR.pl -i sequence.fasta -m 5
	
-IMPORTANT: If the script takes to long to analyze your data you should use FullSSR_sub.pl script. The primer design function in this script
was modified to analyze the portion of the sequence that includes the SSR (2000 pb upstream and downstream of SSR) instead of the whole sequence, in order to improve runtime.
	
	#Execution example: filename big_sequence.fasta and 5 minimun repeats
	$perl FullSSR_sub.pl -i sequence.fasta -m 5	

Results
---------------
When the program finished it will create a folder with the name of 
the sequence and the date and time when the analyse finished.
In this folder you may find a report file (report.html) where you can 
see all the SSRs with the best designed pair of primers for each one (if there are any possible primer).
If you click in any of the ID SSR you will see the microsatellites 
specific information and all the pairs primers designed for it.
Microsatellites and Primer3_results folder contains files with 
information for each SSR and Primers3 original output respectevily.

Questions, bug report, suggestions
---------------
Send us an email:
		juanmanuelcabrera1@gmail.com