SYNOPSIS
RNAmotifAnalysis --fastq seqs.fq --cpus 4 --run
DESCRIPTION
This module pipelines steps in the analysis of SELEX (Systematic Evolution
of Ligands through EXponential enrichment) data.
This main module creates scripts to do the following:
(1) Cluster similar sequences based on edit distance.
(2) Align sequences within each cluster (using mafft).
(3) Calculate the secondary structure of the aligned sequences (using
RNAalifold, from the Vienna RNA package)
(4) Build covariance models using cmbuild from Infernal.
Another useful utility installed with this distribution is
"selex_covarianceSearch" for doing iterative refinements of
covariance models.
If you want to use files that simply list sequences, then use
the "--simple" flag instead of the "--fastq" flag.
This script assumes that you've already done all of the quality
control of your sequences beforehand. If the FASTQ format is
used, quality scores are ignored.
EXAMPLE USE
RNAmotifAnalysis --infile seqs.fq --cpus 4 --run
This will cluster the sequences found in 'seqs.fq' and create a FASTA file
for each one. The FASTA files will be grouped into batches (i.e. one per
cpu requested) that will be placed in a separate directory for each batch,
and processed within that directory. At the end of processing, for each
cluster there will be a covariance model and postscript illustration
files. The batch script used to process each batch will be located in the
respective batch directory. To produce the scripts without running them,
simply exclude the --run flag from the command line.
CONFIGURATION AND ENVIRONMENT
As written, this code makes heavy use of UNIX utilities and is
therefore only supported on UNIX-like environemnts (e.g. Linux, UNIX, Mac
OS X).
Install Infernal, MAFFT, and the RNA Vienna package ahead of time and add
the directories containing their executables to your PATH, so that the
first time you run RNAmotifAnalysis.pm the configuration file (cluster.cfg)
that is generated will have all of the correct parameters. Otherwise,
you'll need to update the configuration file manually.
To update the PATH environment variable with the directory '/usr/local/myapps/bin/',
update your .bashrc file, thus:
echo 'export PATH=/usr/local/myapps/bin:$PATH' >> ~/.bashrc.
Now, every time you open a new terminal window, the PATH environment
variable will contain '/usr/local/myapps/bin/'. To make your new .bashrc
file effective immediately (i.e. without having to open a new terminal
window), use the following command:
source ~/.bashrc
INSTALLATION
These installation instructions assume being able to open and use a
terminal window on Linux.
(0) Some systems need several dependencies installed ahead of time.
You may be able to skip this step. However, if subsequent steps don't
work, then be sure that some basic libraries are installed, as shown
below (or ask a system administrator to take care of it). For the
applicable distribution, open a terminal and then type the commands as
indicated:
For RedHat or CentOS 5.x systems (tested on CentOS 5.5)
sudo yum install gcc
For RedHat or CentOS 6.x systems (tested on "Minimal Desktop" CentOS 6.0)
sudo yum install gcc
sudo yum install perl-devel
For Ubuntu systems (tested on Ubuntu 12-04 LTS)
sudo apt-get install curl
For Debian 5.x systems:
sudo apt-get install gcc
sudo apt-get install make
(1) Install the non-Perl dependencies:
(Versions shown are those that we've tested. Please contact us if
newer versions do not work.)
Infernal 1.0.2 (http://infernal.janelia.org/)
MAFFT 6.849b (http://mafft.cbrc.jp/alignment/software/)
RNA Vienna package 1.8.4 (http://www.tbi.univie.ac.at/~ivo/RNA/)
After installing these, make sure all of the foloowing executables are
in directories within your PATH:
cmbuild
cmcalibrate
cmsearch
cmalign
mafft
RNAalifold
(2) Either (a) download and run our installer or (b) use a CPAN client
to install Bio::App::SELEX::RNAmotifAnalysis.
(a) Installation method: Use the installer.
i. Download installer (and name it "installer")
curl -o installer -L http://ircf.rnet.missouri.edu:8000/share.attachment/200
ii. Make it executable
chmod u+x installer
iii. Run it.
./installer
iv. Delete it if everything installed okay.
rm installer
NOTE: Our installer creates the directory 'perl5' inside your home
directory. This directory serves as the top directory for Perl
modules and executables installed at this time, including
dependencies of the installed modules. The installer also appends
commands to your .bashrc file to make the installed programs
runnable by default (i.e. it includes your local 'perl5/lib/perl5'
directory in the PERL5LIB environment variable and your local
'perl5/bin' directory in your PATH environment variable).
(b) Installation method: Use a CPAN client. Here we demonstrate
the use of cpanminus to install it to a local Perl module
directory. These instructions assume absolutely no experience
with cpanminus.
i. Download cpanminus
curl -LOk http://xrl.us/cpanm
ii. Make it executable
chmod u+x cpanm
iii. Make a local perl5 directory (if it doesn't already exist)
mkdir -p ~/perl5
iv. Add relevant directories to your PERL5LIB and PATH environment
variables by adding the following text to your ~/.bashrc
file:
# Set PERL5LIB if it doesn't already exist
: ${PERL5LIB:=~/perl5/lib/perl5}
# Prepend to PERL5LIB if directory not already found in PERL5LIB
if ! echo $PERL5LIB | egrep -q "(^|:)~/perl5/lib/perl5($|:)"; then
export PERL5LIB=~/perl5/lib/perl5:$PERL5LIB;
fi
# Prepend to PATH if directory not already found in PATH
if ! echo $PATH | egrep -q "(^|:)~/perl5/bin($|:)"; then
export PATH=~/perl5/bin:$PATH;
fi
v. Update environment variables immediately
source ~/.bashrc
vi. Install Module::Build
./cpanm -l ~/perl5 Module::Build
vii. Install Bio::App::SELEX::RNAmotifAnalysis
./cpanm -l ~/perl5 Bio::App::SELEX::RNAmotifAnalysis
Please contact the author if, after consulting this documentation and
searching Google with error messages, you still encounter difficulties
during the installation process using one of these two methods.
INCOMPATIBILITIES
Windows: lacks necessary *nix utilities
SGI: problems with compiled dependency Text::LevenshteinXS
Sun/Solaris: problems with compiled dependency Text::LevenshteinXS
BSD: problems with compiled dependency Text::LevenshteinXS
BUGS AND LIMITATIONS
There are no known bugs in this module.
Please report problems to molecules <at> cpan <dot> org
Patches are welcome.
RELATED PUBLICATIONS
Ditzler MA, Lange MJ, Bose D, Bottoms CA, Virkler KF, et al. (2013) High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase. Nucleic Acids Res 41: 1873–1884. doi: 10.1093/nar/gks1190