Introduction

TOUCAN is a workbench for regulatory sequence analysis on metazoan genomes : comparative genomics, detection of significant transcription factor binding sites, and detection of cis-regulatory modules (combinations of binding sites) in sets of coexpressed/coregulated genes.

It is a platform independent, standalone Java application that is tightly linked with Ensembl, and was built using the BioJava package.SOAP web services are used to remotely access multiple algorithms for comparative genomics, motif detection, and module detection.

Comments, suggestions, and bug reports can be send to stein.aerts<at>med.kuleuven.ac.be or toucan@listserv.cc.kuleuven.ac.be

Register

Launch TOUCAN v. 3.0.2

To run TOUCAN you need to have two things installed:

• Java 2 Platform, Standard Edition (J2SE), version 1.4.x. or 1.5.0
• Java Web Start which is shipped as part of J2SE. From J2SE 1.4.2 onwards it is installed together with the SDK/JRE (more info)

If you have fulfilled these requirements, then you can launch TOUCAN directly using this URL: http://www.esat.kuleuven.ac.be/~saerts/software/toucan.jnlp

Alternatively you can type this command in a terminal window:
javaws http://www.esat.kuleuven.ac.be/~saerts/software/toucan.jnlp

We try to encourage to use Java Web Start because this way you will always have the latest version of the software. This is important because the properties change at least once a month to follow the newest Ensembl release. If you are really unable to use Java Web Start, you can send us an email, and we can give you the JAR file of TOUCAN.

Manual

The manual for release 2 is under construction
The manual of older versions is here. Herbert Mayer has evaluated TOUCAN for Bioinformatics World and has added some useful comments: http://homepage.univie.ac.at/herbert.mayer/MainGEN.html#TOUCAN

Tutorials

• Herbert Mayer's tutorial on the regulatory sequence analysis of genes involved in inflammation: TUTORIAL 1.
  The results of this analysis have been published in: Mayer et al. Deciphering regulatory patterns of inflammatory gene expression from interleukin-1-stimulated human endothelial cells. Arterioscler Thromb Vasc Biol. 2004 Jul;24(7):1192-8.
• Enhancer detection in muscle specific genes, using comparative genomics and module searching: TUTORIAL 2
• Enhancer detection in liver specific genes, using comparative genomics and over-representation statistics (old TOUCAN interface): TUTORIAL 3
• Motif sampling in yeast sequences: TUTORIAL 4
• Analysis of lung-specific genes from the ATLAS expression data: TUTORIAL 5

How to cite?

Stein Aerts, Gert Thijs, Bert Coessens, Mik Staes, Yves Moreau, Bart De Moor (2003) ``TOUCAN : Deciphering the Cis-Regulatory Logic of Coregulated Genes''. Nucl Acids Res, vol. 31, no. 6, 2003, pp. 1753-1764.

Stein Aerts, Peter Van Loo, Gert Thijs, Herbert Mayer, Rainer de Martin, Yves Moreau and Bart De Moor (2005) "TOUCAN 2: the all-inclusive open source workbench for regulatory sequence analysis". Nucl Acids Res, vol. 33 (Web Server issue), W393-6.

NEWS

• The second release of TOUCAN is published: http://nar.oxfordjournals.org/cgi/content/full/33/suppl_2/W393

Source code

TOUCAN is open source software. You can download the client source here and the source for the web services here.

I suggest to communicate via the mailing list, but in case a lot of people would be interested in contributing code, I can make a CVS account or a sourceforge project for TOUCAN.

License

TOUCAN is free software and is distributed under the terms of the GNU General Public License.

Weight matrices

We make several databases of weight matrices available for scoring sequences using either the MotifScanner or the MotifLocator.

• JASPAR
• PlantCARE
• SCPD
• Drosophila collection of Lifanov et al.
• Drosophila compilation: from Halfon et al. and Rajewsky et al.
• Mammalian compilation: from Wasserman & Fickett, Krivan & Wasserman, Frith et al., Bucher, P (1990), Grandori and Eisenman (1997), Kel et al (2001), Mimeault (2000)
• E. coli compilation: Robison et al.
• TRANSFAC 6.0 public.
• The TRANSFAC database is free for users from non-profit organizations only.
• Users from commercial enterprises have to license the TRANSFAC database and accompanying programs.
• Please read the DISCLAIMER

Version

Version 3.0.2
Compilation date: May 2007
Supporting Ensembl release: 42

SOAP Web services

• MotifSampler: Detection of over-represented motifs in the upstream region of coregulated genes
• MotifScanner: Detection of pre-defined motif models (see above) in DNA sequences with background models and probabilistic estimation of the number of hits
• MotifLocator: Detection of pre-defined motif models (see above) in DNA sequences with background models (log odds scores)
• ModuleSearcher: Detection of modules using A* or Genetic Algorithm (combinations of multiple TF-hits)
• AVID/MAVID: alignment program for large genomic regions
• LAGAN/MLAGAN
• BLASTZ
• Footprinter 2.0: phylogenetic footprinting
• If you have suggestions on other algorithms for regulatory sequence analysis that could be useful, please send an email.

Supporting files

• How to construct expected frequency files?
  1) Construct a set of reference regulatory sequences, for example all proximal promoters of the species under study.
  2) Create a background model (choose the order of the Markov model) using the program CreateBackgroundModel, availabe here: http://homes.esat.kuleuven.be/~thijs/download.html
  3) Score these sequences with either MotifScanner or MotifLocator (choose your prior or threshold respectively), both are available here: http://homes.esat.kuleuven.be/~thijs/download.html. You have to use the same backgroundmodel, the same prior/threshold, and the same\a library of motif matrices for the background file as for your actual sequence set. You may contact us to obtain one of the public libraries of motif matrices that ar\e available in TOUCAN.
  4) Convert the GFF output from the scoring to frequencies, using this perl script: GFF2Freq.pl


• If your reference sequence set is small, then you can create a frequency file within TOUCAN: (1) load the reference set; (2) score it with MotifScanner or MotifLocator, choosing a background model and a prior/threshold; (3) choose File->Export->Frequencies.
  
  
• If you would require a specific frequency files but you are unable to execute this procedure, you may contact us.
  
  
• Here are some pre-calculated frequency files:
  • Human
    • Eukaryotic Promoter Database (-499,100 around TSS), MotifScanner, 0th order background model, prior 0.05, TRANSFAC matrices
    • Eukaryotic Promoter Database (-499,100 around TSS), MotifScanner, 0th order background model, prior 0.1, TRANSFAC matrices
    • Eukaryotic Promoter Database (-499,100 around TSS), MotifScanner, 0th order background model, prior 0.2, TRANSFAC matrices
    • Eukaryotic Promoter Database (-499,100 around TSS), MotifLocator, 0th order background model, threshold 0.9, TRANSFAC matrices
    • Human-Mouse conserved regions within 10kb upstream sequence (Lagan,75% identity,minimum 100bp), MotifScanner, 0th order background model, prior 0.2, TRANSFAC matrices
  • Mouse
  • 1000 randomly selected genes from Ensembl, 1kb proximal sequence, MotifScanner, 0th order background model, prior 0.2
  • 1000 randomly selected genes from Ensembl, 1kb proximal sequence, MotifLocator, 0th order background model, threshold 0.9
  • Drosophila
    • Eukaryotic Promoter Database (-499,100 around TSS), MotifScanner, 0th order background model, prior 0.2
    • Eukaryotic Promoter Database (-499,100 around TSS), MotifLocator, 0th order background model, threshold 0.9

Mailing list

If you're downloading TOUCAN, be aware that it is a beta-version. It is constantly being updated and improved. Therefore you can subscribe to the TOUCAN mailinglist either by clicking here, or by sending an e-mail to LISTSERV@LISTSERV.CC.KULEUVEN.AC.BE with only "subscribe toucan" in the message body.
This way we can notify you when a new version is available, when bugs are fixed, or when new features are added. You can also send e-mails with questions and feedback to the developers and to other users: toucan@listserv.cc.kuleuven.ac.be

Main features

• Retrieve (upstream) sequences and annotation from Ensembl and EMBL automatically
• Retrieve (upstream) sequences of one or more ortologous genes automatically
• Visualize a sequence set with all annotated features
• Add annotation in GFF format
• CpG island annotation
• Align large genomic regions with various specialized algorithms like AVID, LAGAN, or BLASTZ
• Easily select potentially interesting regulatory regions, like conserved non-coding sequences (CNS) and proximal promoters
• Predict instances of known transcription factors using the MotifScanner algorithm (with higher-order background models), with libraries of position weight matrices (e.g., TRANSFAC, JASPAR, ...)
• Detect over-represented instances of these factors using a binomial distribution model
• Detect over-represented motifs using the MotifSampler implementation of the Gibbs sampler, with higher-order background models
• Detect cis-regulatory modules using the ModuleSearcher web service: the best combination of sites in your set
• Import/export in standard formats (EMBL, fastA, Genbank), and as JPG or PNG images.

Related tools

• Tools for regulatory sequence analysis:
  • ConSite
  • rVISTA
  • RSAT
  • TRAFAC
  • SeqVista
• Tools for genome-wide cis-regulatory module detection
  • MSCAN
  • CREME
  • Stubb
  • MCAST
  • Ahab,/a>
  • Cluster-buster
  • PFRSearch
• Comparative genomics tools
  • AVID + VISTA
  • Blastz + PipMaker
  • LAGAN

Service mirrors

If you wish to install the TOUCAN services locally, and make them available to the community (thereby increasing the overall uptime of the services), you can send me an email to obtain the source code of the services and the installation instructions.

Screenshots

Acknowledgements

We wish to thank all the people and research groups who made their data or software available.

Please cite the services you've used within TOUCAN:

ENSEMBL

• Birney E, Andrews TD, Bevan P, Caccamo M, Chen Y, Clarke L, Coates G, Cuff J, Curwen V, Cutts T, Down T, Eyras E, Fernandez-Suarez XM, Gane P, Gibbins B, Gilbert J, Hammond M, Hotz HR, Iyer V, Jekosch K, Kahari A, Kasprzyk A, Keefe D, Keenan S, Lehvaslaiho H, McVicker G, Melsopp C, Meidl P, Mongin E, Pettett R, Potter S, Proctor G, Rae M, Searle S, Slater G, Smedley D, Smith J, Spooner W, Stabenau A, Stalker J, Storey R, Ureta-Vidal A, Woodwark KC, Cameron G, Durbin R, Cox A, Hubbard T, Clamp M. An overview of Ensembl. Genome Res. 2004 May;14(5):925-8. Epub 2004 Apr 12. Review.

TRANSFAC

• Wingender E, Chen X, Fricke E, Geffers R, Hehl R, Liebich I, Krull M, Matys V, Michael H, Ohnhauser R, Pruss M, Schacherer F, Thiele S, Urbach S. The TRANSFAC system on gene expression regulation. Nucleic Acids Res. 2001 Jan 1;29(1):281-3.
Note:The TRANSFAC database is free for users from non-profit organizations only. Users from commercial enterprises have to license the TRANSFAC database and accompanying programs. Please read the DISCLAIMER at http://www.gene-regulation.com/pub/databases/transfac/disclaimer.html.

JASPAR

• Sandelin A, Alkema W, Engstrom P, Wasserman WW, Lenhard B. JASPAR: an open-access database for eukaryotic transcription factor binding profiles. Nucleic Acids Res. 2004 Jan 1;32 Database issue:D91-4.

SCPD

• Zhu J, Zhang MQ. SCPD: a promoter database of the yeast Saccharomyces cerevisiae. Bioinformatics. 1999 Jul-Aug;15(7-8):607-11.

PLANTCARE

• Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002 Jan 1;30(1):325-7.

AVID, MAVID, VISTA

• Bray N, Dubchak I, Pachter L. AVID: A global alignment program. Genome Res. 2003 Jan;13(1):97-102.
• Mayor C, Brudno M, Schwartz JR, Poliakov A, Rubin EM, Frazer KA, Pachter LS, Dubchak I. VISTA : visualizing global DNA sequence alignments of arbitrary length. Bioinformatics. 2000 Nov;16(11):1046-7.
• Bray N, Pachter L. MAVID multiple alignment server. Nucleic Acids Res. 2003 Jul 1;31(13):3525-6.

LAGAN, MLAGAN

• Brudno M, Do CB, Cooper GM, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S; NISC Comparative Sequencing Program. LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA. Genome Res. 2003 Apr;13(4):721-31. Epub 2003 Mar 12.

BLASTZ, PIPMAKER

• Schwartz S, Kent WJ, Smit A, Zhang Z, Baertsch R, Hardison RC, Haussler D, Miller W. Human-mouse alignments with BLASTZ. Genome Res. 2003 Jan;13(1):103-7. Erratum in: Genome Res. 2004 Apr;14(4):786.
• Schwartz S, Zhang Z, Frazer KA, Smit A, Riemer C, Bouck J, Gibbs R, Hardison R, Miller W. PipMaker--a web server for aligning two genomic DNA sequences. Genome Res. 2000 Apr;10(4):577-86.

MOTIFSCANNER

• Aerts S, Thijs G, Coessens B, Staes M, Moreau Y, De Moor B. Toucan: deciphering the cis-regulatory logic of coregulated genes. Nucleic Acids Res. 2003 Mar 15;31(6):1753-64.

MOTIFSAMPLER

• Thijs G, Marchal K, Lescot M, Rombauts S, De Moor B, Rouze P, Moreau Y. A Gibbs sampling method to detect overrepresented motifs in the upstream regions of coexpressed genes. J Comput Biol. 2002;9(2):447-64.
• Thijs G, Lescot M, Marchal K, Rombauts S, De Moor B, Rouze P, Moreau Y. A higher-order background model improves the detection of promoter regulatory elements by Gibbs sampling.

MODULESEARCHER

• Aerts S, Van Loo P, Thijs G, Moreau Y, De Moor B. Computational detection of cis -regulatory modules. Bioinformatics. 2003 Oct;19 Suppl 2:II5-II14.
• Aerts S, Van Loo P, Moreau Y, De Moor B. A genetic algorithm for the detection of new cis-regulatory modules in sets of coregulated genes. Bioinformatics. 2004 Aug 12;20(12):1974-6. Epub 2004 Mar 25.

FOOTPRINTER

• Blanchette M, Tompa M. Discovery of regulatory elements by a computational method for phylogenetic footprinting. Genome Res. 2002 May;12(5):739-48.
• Blanchette M, Schwikowski B, Tompa M. Algorithms for phylogenetic footprinting. J Comput Biol. 2002;9(2):211-23.