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MetNet Plugins: Getting started with Cytoscape

1. Introduction The MetNet Plugins are Cytoscape plugins (cytoscape2.4 and 2.5) dedicated to providing useful visualization and an integrated analysis tools for metabolic networks. However, to make full use of these tools, the user must have basic knowledge of loading networks/laying out networks/controlling visual preferences such as color and node size. General purpose tutorials for these activities can be downloaded at www.cytoscape.org. The explanations given here focus on networks from the MetNetDB database, although they should work for networks from other sources as well

1.1 Structure of this Document Section 1. Information on getting started with plugins and tutorial. Section 2. Tutorial for loading networks into Cytoscape using session files. Section 3. Tutorial on loading networks from MetNetDB and laying out graph networks in

Cytoscape. Section 4. Visual preference control in Cytoscape.

1.2 Loading the MetNet Plugins Before starting the tutorial, please make sure that you have downloaded and installed a current version of Cytoscape (2.4 or higher) on your computer from www.cytoscape.org. Next, download the Subgraphcreator plugin from the MetNet for Cytoscape plugin page. Put the file, Subgraphcreator1.x.jar, into the folder called plugins in the Cytoscape directory on your computer. The directory on a Windows computer is shown in Figure 1.

Figure 1 Location of the plugins and sampleData folders in the Cytoscape application.

1.3 Loading the MetNet Sample Data Files Before starting the tutorial, please make sure that you have downloaded and installed the MetNet sample data files from the MetNet plugin page. Unzip the file, MetNetsampledata.zip, into the folder

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called sampleData in the Cytoscape directory on your computer for this tutorial. The sampleData directory on a Windows computer is shown in Figure 1. The tutorial assumes that the MetNet Sample data is in the Cytoscape sampleData folder. Once the plugin and data have been added to the proper folders, open the Cytoscape application.

2. Loading and Manipulating a Network in Cytoscape 1. Complete the instructions in sections 1.2 and 1.3 above before starting the tutorial to make sure that the plugin and sample data are properly installed. The tutorial assumes that the MetNet Sample data are in the Cytoscape sampleData folder. Start Cytoscape by clicking on the icon for the Cytoscape.exe file. The main Cytoscape window should open and look like Figure 2 below.

Figure 2. Main Cytoscape Window. The window is divided into three panels. The upper right is the drawing window where the graph network will be drawn. The lower right is the Data Panel which gives information about the network nodes. The left panel is the control panel which has different tabs for different functionalities. 2. Open the Cytoscape session file, Acetyl-CoAbiotin.cys by selecting File-> Open on the top Cytoscape menu and selecting the file. This session file contains a pathway file, an associated visual mapping file, and information on what graph layout to use. Your screen should look like Figure 3.

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Figure 3. Selection window for the Cytoscape session file. 3. After opening the Cytoscape session file, Acetyl-CoAbiotin.cys your window should look like Figure 4. This session file contains a pathway file, an associated visual mapping file, and information on what graph layout to use. If you are not familiar with Cytoscape, explore the network by zooming in using the magnifying glass icon. Also, try selecting alternative graph layouts on the Layout menu at the top. For more details on the basic use of Cytoscape, please look at the online tutorials available at the Cytoscape website. Information about individual nodes can be listed in the Data Panel in the lower right hand corner of the Cytoscape window. Right clicking on the icon circled in red in Figure 5 allows the user to select what node attributes can be seen. For this tutorial, right click on the icon and select the location, nodeType, and show boxes. This gives information about the subcellular location, the role of the node, and the name of the node. Try highlighting a few nodes (they will turn pink) and looking at the information as shown in Figure 5. Important note: Network data from different sources will have different attributes and names. This information only holds for data from the MetNetDB Arabidopsis pathway database.

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Figure 4. Cytoscape window upon opening the Acetyl-CoAbiotin.cys file. This session file contains pathway data, visual attributes for the graph, and graph layout information. Clicking on the tab labeled VizMapper in the Control Panel will allow exploration of the node colors.

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Figure 5. Information about the selected nodes, highlighted in pink, is displayed in the Data Panel in the lower left corner. Right click on the circled icon to choose which attribute fields are shown. 4. “Link-out” capability. For many nodes in a network, there is quite a quite a bit of additional

annotation available at other websites. As long as the nodes use common nomenclature for naming, Cytoscape users can access other data sources for gene and protein annotation. To access this capability, select a node and right click on it. This will bring up the menu shown in Figure 6 below. Select the option “Link out” and a data source to open a web browser and search for the desired term. An example of such a web search is shown in Figure 7. This function can be customized to access other databases as well. Instructions can be found in the Cytoscape manual.

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Figure 6. Right click on a node to get additional information using the Link out functionality. In this case, NCBI Entrez Gene is selected.

Figure 7. The results of the Link Out search for a gene, AT1G52670.

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3. Using MetNet Network files in Cytoscape Note: Network data files from the MetNetDB project can be generated using the XML Builder application at http://metnet.vrac.iastate.edu/MetNet_fcmodeler.htm. Click the option XML Builder on the menu bar to access. This application produces pathway files in the MetNet XML format from the MetNetDB database.

1. To open a network file that is not in the Cytoscape session format, Select the option File-> Import -> Network (multiple file types) as shown in Figure 8. The window shown in Figure 9 will come up. Usually the files will be stored on your local machine. Use the Select option to choose a file.

Figure 8. Selecting the Import –> Network (multiple file types) option.

Figure 9. Import Network file window. Click select to choose a file.

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2. For this tutorial, select the file Acetyl-CoA Biotin network (Expert User).xml as shown in Figure 10. Then click the import button as shown in Figure 11. A status window will come up to let you know that the network is loading and the size of the network. Click close in the status window to go onto the next step.

Figure 10. Find the MetNetSampledata directory and select the file Acetyl-CoA Biotin network (Expert User).xml. Then click on the Open button.

Figure 11. After selecting the file, click the Import Button.

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3. Laying out the network: After loading the network, the window will look as shown in Figure 12 . As this is clearly not useful, the next step is to apply a Layout algorithm to the network. Cytoscape contains a number of algorithms for layouts. The best layout depends on the structure and complexity of your network and what problem you are trying to answer. Select Layout on the top menu of Cytoscape as shown in Figure 12. Select the option yFiles and then the layout “Organic”. Figure 13 shows the network after the layout algorithm is applied. Experiment with other layout algorithms to find structures that are appealing. Also, zoom in using the magnifying glass icon or by holding down the right mouse button and moving your mouse. The next step in creating a usable picture is to choose visual mapping properties for your network.

Figure 12 Cytoscape window after loading the XML pathway network. The Layout menu item is selected. Choose the yFiles option and select the Organic layout.

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Figure 13. The Acetyl CoA Biotin pathway laid out with the yFiles Organic layout.

4. Using MetNet Visual Styles in Cytoscape The final step to creating a usable network view in Cytoscape is to apply a visual style to your network. Visual styles control the colors and sizes of nodes and edges as well as what is written inside a node or by an edge. This allows you to have many different looks for the same network depending on whether you want to focus on different organelles or node types, etc. 1. Opening a Vizmap Property File. Vizmap property files are imported into Cytoscape just like network files. We will now load the MetNetStyle Vizmap properties file, called MetNetStyle.props. Go to the File Menu and select: File -> Import -> Vizmap Property File as shown in Figure 14. Visual style files have the suffix .props. Select the file MetNetStyle.props as shown in Figure 15. Figure 16 shows the resulting status window. Close the window when the file has loaded.

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Figure 14. Importing a Vizmap property file.

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Figure 15. Select the MetNetStyle.props file from the MetNetSampleData directory on your computer. Click the Open button.

Figure 16. A status window will show that the file is being imported into Cytoscape. Click the Close button when loading is completed.

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2. Applying the visual style. Once the Vizmap has loaded, it needs to be applied to the network being displayed. Select the Vizmapper tab (Shown circled in Figure 17). In the Vizmapper tab, there is a field by the top labeled current visual style (Shown circled in Figure 18), make sure that this field is set to MetNetStyle. The network display should look much more colorful. Try zooming in to the graph using the magnifying glass icon or by holding down the right mouse button and moving the mouse.

Figure 17. The Cytoscape window after loading the Vizmap style MetNetStyle.props. Click the circled VizMapper tab to learn more about this style

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Figure 18. Zoom in on the graph to see the colors and labels with the MetNetStyle. The VizMapper tab on the left gives information about the style and mappings. The style name is circled at the top of this tab. 3. Getting a Legend for a Visual Style. Right clicking the editing icon in the circle in Figure 18, will give a menu for adding, deleting, or copying a visual style. The last option will create a legend for the existing style as shown in Figure 19.

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Figure 19. Legend for MetNetStyle.

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4. Saving your work. After all the work involved in setting up a file for visualization and analysis, it is important to save the current layout and visual style in a Cytoscape session for future work. Select the File menu -> Save will save your current session, including your preferred layout and color scheme.

Figure 20. Saving your current session for future use.