PREREQUISITES Perl v5.10 is recommended, at least Perl v5.8 is required, although not everything has been tested with the older version. Required perl libraries are Bio::BioStudio and Bio::GeneDesign, which are available from cpan $ sudo cpan > install Bio::BioStudio::Basic Bio::GeneDesign::Basic or from bitbucket $ git clone https://bitbucket.org/notadoctor/biostudio-lib.git $ git clone https://bitbucket.org/notadoctor/genedesign-lib.git $ cd [biostudio/genedesign]-lib $ sudo perl Build.PL $ sudo perl ./Build test $ sudo perl ./Build install MySQL 5 is used by BioStudio to index genomes, and by GBrowse2 as a data storage engine. WARNING! If you use 64 bit perl, use 64 bit mysql. Mixing 64 bit and 32 bit mysql/perl will result in an intractable library error from DBD::mysql and DBI. You will need to create a `bss' user for BioStudio to use MySQL; $ mysql -u root > create user 'bss'@'localhost' identified by 'bsspass'; > grant SELECT,INSERT,UPDATE,DELETE,CREATE,DROP to 'bss'@'localhost'; BLAST+ is required for chromosome comparison and PCR Tag generation. Use the new blast+ executabes, available from ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ and not the older blast you compile in C yourself. You can tell you have the right one by its inclusion of the legacy_blast.pl utility. If you use the Mac OS X .dmg installer, you may have to correct the legacy_blast.pl script; change line 43 to read use constant DEFAULT_PATH => "/usr/local/ncbi/blast/bin"; or whatever directory you installed BLAST+ to and it should be fine. GBrowse is not required by BioStudio, but it currently provides the only graphical user interface. If you do not want to use BioStudio on the command line, you will have to use GBrowse. Here's how to install GBrowse. You need a webserver; I recommend Apache 2; GBrowse2 will automatically configure Apache and it is often already on the computer you are using. Graphics libraries: libjpeg http://sourceforge.net/projects/libjpeg/files/ libpng http://sourceforge.net/projects/libpng/files/ libgd https://bitbucket.org/pierrejoye/gd-libgd Bioperl-live is required; this is the development branch. The stable will not do for GBrowse2! $ git clone git://github.com/bioperl/bioperl-live.git $ cd bioperl-live/ $ sudo perl Build.PL $ sudo ./Build installdeps $ sudo ./Build test $ sudo ./Build install GBrowse2 is available from github, the defaults are usually appropriate. $ git clone git://github.com/GMOD/GBrowse.git $ cd Generic-Genome-Browser/ $ sudo perl Build.PL $ sudo ./Build installdeps $ sudo ./Build test $ sudo ./Build config $ sudo ./Build install INSTALLATION To install BioStudio, run the following command: sudo perl DEPLOY.PL GETTING STARTED Here's an example with yeast. We must fetch the latest S288C genome annotation from SGD in gff3 format. http://downloads.yeastgenome.org/chromosomal_feature/saccharomyces_cerevisiae.gff This file includes all sixteen chromosomes, the mitochondrial chromosome, and the 2-micron plasmid, making it large and too unwieldy to be useful. I usually append the accession date to the SGD filename, _YYMMDD, so that the age of the annotation will be included in the new gff3 files. We will split the SGD file into separate gff3 files for each DNA molecule with the BS_GFF3Split.pl utility. SGD does not include information about essentiality or fast growth phenotype in the GFF3 file (it may be in the Note tag, but nowhere else), but BioStudio uses that information to render gene features in attention grabbing colors and to make decisions about edit preceence. BS_GFF3_Split.pl will take lists of essential genes and a list of genes required for fast growth, and annotate the newly split chromosomes with that information. BS_GFF3Split.pl -F saccharomyces_cerevisiae.gff -S yeast -L essentials.txt Now the genome repository is populated with 16 chromosomes. To get a chromosome into GBrowse, we run the BS_PrepareGBrowse.pl script: BS_PrepareGBrowse -CHR yeast_chr01_0_00 Now you can navigate to localhost/cgi-bin/gb2/gbrowse/yeast_chr01_0_00 and begin graphically editing the chromosome. TROUBLESHOOTING Some of GBrowse2’s default features can obscure the editing process. For instance, there is a new “padding” option that shows flanking data on either side of a view; this may be confusing to anyone who used the original GBrowse and is used to assuming that what is in view is actually encompassed by the current coordinates. To avoid this problem, edit GBrowse.conf and set “pad_left = 0” and “pad_right = 0”. Also, GBrowse2 has a “details multiplier”, which loads extra data so a user can scroll right or left in the details view. This can be a problem if a user accidentally shifts the view without realizing that they are changing the orders to the editor; they will not be modifying the segment they think they are modifying. If this seems to be a problem, set “details multiplier = 0”. Make sure that the genome repository folder, the BLAST folder, the tmp folder, and the bs_confs folder are world writeable (substitute your paths): chmod -R 777 /etc/BioStudio/genomes chmod -R 777 /etc/BioStudio/blast chmod -R 777 /etc/BioStudio/tmp chmod -R 777 /etc/gbrowse2/bs_confs Make sure the BioStudio bin directory is full of executables: chmod -R 755 /usr/local/BioStudio/bin SUPPORT AND DOCUMENTATION Each script has POD documentation embedded; documentation is ongoing. Email firstname.lastname@example.org for support. LICENSE AND COPYRIGHT Copyright (C) 2011 Sarah Richardson This program is distributed under the (Revised) BSD License: L<http://www.opensource.org/licenses/bsd-license.php> Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met: * Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer. * Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution. * Neither the name of Sarah Richardson's Organization nor the names of its contributors may be used to endorse or promote products derived from this software without specific prior written permission. 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