<noautolink> ---+ Predicting Riboswitch Regulation on a Genomic Scale This page describes how to install a set of Perl scripts designed to identify members of known [[http://en.wikipedia.org/wiki/Riboswitch][riboswitch classes]] in genomic sequences. It accompanies a _[[http://www.springer.com/humana+press/biochemistry/book/978-1-934115-88-6][Methods in Molecular Biology]]_ protocol chapter that explains how to use these scripts. This protocol may also be useful for analyzing other structured RNAs, such as those found in the [[http://www.sanger.ac.uk/Software/Rfam/][Rfam database]], or for investigating new regulatory RNA motifs, such as might be predicted by [[http://bio.cs.washington.edu/yzizhen/CMfinder/][CMfinder]]. <div style="background-color:rgb(255,200,200);margin:10px; padding:10px;"><table><tr><td width=88><img src="%PUBURL%/%WEB%/SharedImages/wip.gif"></td> <td valign="center"> *Please note:* Infernal and BioPerl have changed since this protocol was published, so the directions here differ somewhat from those in the book chapter. They are currently being updated. %MAKETEXT{"If you have any feedback, please [[[_1]][let me know]]." args="mailto:%WIKIWEBMASTER%"}% See the [[SupplementRiboswitchPrediction_v1][original installation instructions (v1)]]. </td></tr></table></div> ---++ Installation ---+++ Infernal [[http://infernal.janelia.org/][Infernal]] is used to conduct covariance model searches. Download, compile, and install according to the directions for your system. There have been some interface changes since v0.81. This updated protocol assumes you have v1.0. ---+++ <nop>BioPerl As of June 2009, the current stable release of <nop>BioPerl (v1.6) does not include the necessary modules for parsing Infernal search results or manipulating Stockholm alignments. Therefore, you may need to download and install the current CVS version of *bioperl-live*. Either follow the [[http://www.bioperl.org/wiki/Getting_BioPerl][Getting BioPerl]] instructions or try this link to [[http://code.open-bio.org/cgi/viewcvs.cgi/bioperl-live/bioperl-live.tar.gz?tarball=1][download directly]]. ---+++ Code Libraries [[http://www.libgd.org/Main_Page][libGD]] is required to produce drawings of the genomic contexts of putative RNA structure matches. Download and install according to the directions for your system. ---+++ Perl Modules Several additional Perl modules from are required for the riboswitch perl scripts. You can download and install them directly from [[http://www.cpan.org/][CPAN]]. Alternately, on MacOS X, you can use the following command sequence if you have administrator privileges: <code><div style="border-color: grey; border-style: solid; border-width: 1px; padding:1px;"> >sudo perl -MCPAN -e shell<br> >Password: ********<br> >install Storable<br> >install GD<br> </div></code> The GD module requires libGD to be installed beforehand and is only necessary for producing genomic context drawings. If you encounter any other messages of the form: "Can't locate <code>module.pm</code> in @INC", try installing <code>module</code> from CPAN. ---+++ Riboswitch Perl Scripts Download the riboswitch Perl scripts. | [[%ATTACHURL%/riboswitch_methods_v1.tar.gz][%ICON{download}%]] | version 1 | 6 January 2008 | ---++ Usage Each Perl script has POD documentation. To view it, either open the script in a text editor or use one of these commands: <code><div style="border-color: grey; border-style: solid; border-width: 1px; padding:1px;"> >perldoc genome_context.pl<br> >genome_context.pl --man</div></code> ---++ Updated Protocols ---+++ 3.2 Annotating Riboswitches in a Bacterial Genome With the new version of Infernal (v1.0), the command sequence for performing global alignment of the riboswitch model to matches in the database with the hmmfilter has changed as follows: <code><div style="border-color: grey; border-style: solid; border-width: 1px; padding:1px;"> >cmbuild TPP.cm TPP.sto<br> >cmsearch -g -T 27.2 TPP.cm NC_009832.1.fna > hits.txt<br> >cmsearch_reformat.pl -s scores.tab hits.txt hits.fna<br> >cmalign -o hits.sto TPP.cm hits.fna<br> >stockholm_to_html.pl hits.sto hits.html<br> >genomic_context.pl -b scores.tab -s NC_009832.1.gbk hits.sto context </div></code> ---+++ 3.3 Exhaustively Identifying Riboswitches in a Sequence Database With the new version of Infernal (v1.0), the example command sequence for performing global alignment of the riboswitch model to matches in the database has changed as follows: <code><div style="border-color: grey; border-style: solid; border-width: 1px; padding:1px;"> >fragment_db.pl -s 40 /db/refseq24/fasta /db/refseq24/fragment<br> >cmbuild TPP.cm TPP.sto<br> >cmcalibrate TPP.cm<br> >cmsearch -g TPP.cm /db/refseq24/fragment/1.fna > hits.1.txt<br> >cat hits.* > hits.txt<br> >cmsearch_reformat.pl -T 10 -b scores.tab hits.txt hits.fna<br> >cmalign -o hits.sto TPP.cm hits.fna<br> >stockholm_to_html.pl hits.sto hits.html<br> >index_db.pl -d refseq24_genbank /db/refseq24/genbank /db/index<br> >genomic_context.pl -b scores.tab -d /db/index/refseq24 hits.sto context<br> </div></code> Especially note the additional <code>cmcalibrate</code> step which accelerates the <code>cmsearch</code>.
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