= R2 and Cytoscape: Inferring gene targets by graph based integration and analysis of molecular biological data =
'''Piet Molenaar'''

''Academic Medical Center, University of Amsterdam, the Netherlands''

'''__Biological Use Case__''': In our lab we're doing research into the molecular networks involved in childhood cancer. To this end we've profiled a large set of childhood tumors. Guided by biological questions we set out to develop a web-based array analysis platform called R2. In order to make an educated guess what genes to test that appeared of importance in this analysis we needed additional network analysis.

'''__Recipe__'''

''__Cytoscape version__'': 2.4 - 2.6 

''__Plugins to Load__'': R2Plugin, [[http://baderlab.org/Software/NetMatch|NetMatch]]

''__GUI steps__'': 

|| ''Story'' || ''Action'' || ''Remarks'' ||
|| 1. Research into childhood tumors. Array data analyzed using web-based R2. Biologists use tumor series to extract relevant genes. These are manipulated in tumor cell-lines using siRna and studied in time-series || Open website: http://hgserver1.amc.nl/cgi-bin/r2/main.cgi ||  ||
|| 2. Access and datasets input still restricted; will change. For logon mail r2-support_at_amc.uva.nl || Logon with: un pw || If problem use the session file below (Piet: todo provide restricted access logon) ||
|| 3. R2 portal shows; menu reflects all possibilities, lots of data (>17,000 arrays), clinical data, all types of stat analysis || Type MYCN ||  ||
|| 4. Note based on  || Click timeseries ||  ||
|| 5. Panel provides you with three choices: __Explore timeseries__: single gene in series; __TableBuilder__: Find genes in series based on tresholds; __Multiplo__: Limma statistics applied to replicates. Availability of other datasets under dropdown  || Select TableBuilder || Keep default selection set_hg_mix, upcoming improved naming of these sets ||
|| 6. Tree reflects the cell-lines || Choose cellline imr ||  ||
|| 7. Under cell-line the manipulated genes and type of manipulation; multiple selection possible || Choose Notch1 || Multiple selection is tricky! ||
|| 8. There are several cut-offs and tresholds to set, hugoonce, guarantees only one hugo per probeset; create networks, creates the cytoscape network ||Set hugoonce, create networks, use manipulated gene to yes. Set cutoff to 100, logfold to 1  ||  ||
|| 9. List of regulated genes is created. Now create a network || Click on button || If not running webstart is created ||
||10. We selected treat gene as node so it is treated as network, Note time points layout ||  ||  ||
||11. Now gather extra knowledge; protein notch3 upregulated, what influence has this on the transcription factor regulatory network, ie: what tf regulate targets downstream in time and have binding partners present; Hard for biologist typical computational task || Click Plugins->TF partner analysis->Add time causal network || If people want to: show code, note att names ||
||12. Do some vizmapping  ||Create vizmapping edge color for Tf-interaction attribute; has two discrete values  || Binding partners red, TF sites green ||
||13. Do some filtering  || Create greater 3 neighbours filter, select and deselect source node... ||  ||
|| 14. Open netmatch; netmatch has a graph query facility enabling you to draw a query including attribute values || Click the pencil icon in the top-bar of the netmatch panel ||  ||
|| 15. Create / load query network  || Draw a network three nodes; one in the middle having two edges; Add att values by right clicking on edges: 'NCBI-TF-Interaction' any direction; 'Transcription factor binding sites' outgoing. Click on the 'pass query' button|| Query network inc atts can be downloaded below ||
|| 16. Now the query is performed   || Set att names for nodes to id, for edges to Tf-Interaction ||  ||
|| 17. In the results panel the resulting subnetworks appear  || Enable the create subnetwork box, click on one of the result networks, set vizmap to timeseries in vizmapper ||  ||
|| 18. We now have a biological relevant network of a TF having a binding partner present and regulating a target downstream  ||  || Note that additional logic can be incorporated; up/down etc  ||

'''__Data Files__''': Supplied via website R2. Sample session file: [[attachment:Notch3ICFiltered.cys|Notch3ICFiltered]]
Netmatch query files: [[attachment:NetmatchQuery.EA|EdgeAtts]], [[attachment:NetmatchQuery.NA|NodeAtts]], [[attachment:NetmatchQuery.sif|NetmatchQuery sif file]]

'''__Presentation__''': [[attachment:PietMolenaar_ICSB_23_8_08.ppt|R2AndCytoscape_PPT]]

'''__Webstart__''': Time permitting

'''__Video__''': Time permitting