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 <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