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) 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 cpan 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