NAME
Bio::App::SELEX::RNAmotifAnalysis - Cluster SELEX sequences and calculate their structures
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.
The output file contains names that contain four period delimited values
For example, 2.3.1.5 means
that this is the second cluster
this is the third sequence in the cluster
there is one copy of this sequences
it is an edit distance of 5 from the reference sequence
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) Use a CPAN client to install Bio::App::SELEX::RNAmotifAnalysis.
Here we demonstrate the use of cpanminus to install it to a local Perl module directory. These instructions assume absolutely no experience with cpanminus.
1. Download cpanminus
curl -LOk http://xrl.us/cpanm
2. Make it executable
chmod u+x cpanm
3. Make a local lib/perl5 directory (if it doesn't already exist)
mkdir -p ~/lib/perl5
4. 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:=~/lib/perl5}
# Prepend to PERL5LIB if directory not already found in PERL5LIB
if ! echo $PERL5LIB | egrep -q "(^|:)~/perl5/lib/perl5($|:)"; then
export PERL5LIB=~/lib/perl5:$PERL5LIB;
fi
# Prepend to PATH if directory not already found in PATH
if ! echo $PATH | egrep -q "(^|:)~/perl5/bin($|:)"; then
export PATH=~/bin:$PATH;
fi
5. Update environment variables immediately
source ~/.bashrc
6. Install Module::Build
./cpanm Module::Build
7. Install Text::LevenshteinXS (even if you already have it installed elsewhere)
./cpanm Text::LevenshteinXS
8. Install Bio::App::SELEX::RNAmotifAnalysis
./cpanm 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.
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.
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(3):1873-1884. doi: 10.1093/nar/gks1190