Difference: SupplementRiboswitchPrediction (3 vs. 4)

Revision 42009-06-23 - JeffreyBarrick

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Predicting Riboswitch Regulation on a Genomic Scale

This page describes how to install a set of Perl scripts designed to identify members of known riboswitch classes in genomic sequences. It accompanies a 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 Rfam database, or for investigating new regulatory RNA motifs, such as might be predicted by CMfinder.

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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. If you have any feedback, please let me know. See the original installation instructions (v1).

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Please note: Infernal and BioPerl have changed since this protocol was published, so the directions here differ somewhat from those in the book chapter. If you have any feedback, please let me know. You can also visit the original installation instructions (v1).

Installation

Infernal

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.

BioPerl

As of June 2009, the current stable release of 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 Getting BioPerl instructions or try this link to download directly.

Code Libraries

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 CPAN. Alternately, on MacOS X, you can use the following command sequence if you have administrator privileges:


 >sudo perl -MCPAN -e shell

 >Password: ********

 >install Storable

 >install GD

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> >install Bio::Graphics

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 module.pm in @INC", try installing module from CPAN.

Riboswitch Perl Scripts

Download the riboswitch Perl scripts.

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version 1

6 January 2008

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version 2

23 June 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:

 >perldoc genome_context.pl
 >genome_context.pl --man

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:


 >cmbuild TPP.cm TPP.sto

 >cmsearch -g  -T 27.2 TPP.cm NC_009832.1.fna > hits.txt

 >cmsearch_reformat.pl -s scores.tab hits.txt hits.fna

 >cmalign -o hits.sto TPP.cm hits.fna

 >stockholm_to_html.pl hits.sto hits.html

 >genomic_context.pl -b scores.tab -s NC_009832.1.gbk hits.sto context

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:


 >fragment_db.pl -s 40 /db/refseq24/fasta /db/refseq24/fragment

 >cmbuild TPP.cm TPP.sto

 >cmsearch -g TPP.cm /db/refseq24/fragment/1.fna > hits.1.txt

 >cat hits.* > hits.txt

 >cmsearch_reformat.pl -T 10 -b scores.tab hits.txt hits.fna

 >cmalign -o hits.sto TPP.cm hits.fna

 >stockholm_to_html.pl hits.sto hits.html

 >index_db.pl -d refseq24_genbank /db/refseq24/genbank /db/index

 >genomic_context.pl -b scores.tab -d /db/index/refseq24 hits.sto context

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