Network Analysis, Visualization, & Graphing TORonto (NAViGaTOR) User Documentation

Transcription

1 Network Analysis, Visualization, & Graphing TORonto (NAViGaTOR) User Documentation Jurisica Lab, Ontario Cancer Institute November 10, 2006 Contents 1 Introduction 2 2 Installation 2 3 Getting Started Force-Directed Layout File Formats Tab-Delimited Text File Navigator XML Format PSI-MI Format Ontology, Expression levels, and Interaction Strengths User Interface Functionality Application Menu File Menu Select Menu View Menu Export Menu Layout Menu Analysis Menu Ophid Menu D Options Menu Application Tool Bar Application Control Panel Description Panel Right-Click Menu Mouse and Keyboard Controls Additional Resources Tutorials

2 1 Introduction Network Analysis, Visualization, & Graphing TORonto (NAViGaTOR) is a software package for visualizing and analyzing protein-protein interaction networks. In these networks, nodes represent the interactors (proteins, or their associated genes), and edges represent interactions. NAViGaTOR provides a framework on which molecular information related to protein interactions can be visualized. Figure 1: NAViGaTOR screenshot. The snapshot in Figure 1 gives a sample view of a protein-protein interaction network. NAViGaTOR uses a colour scheme to code information such as protein ontology, expression levels, significant interactions, and interaction strength. For example, in Figure 1, the colours of nodes indicate categories in an ontology described in the input file. Also, the edge colours in this graph are based on significant interactions occurring in the two groups of data that are loaded into the network. For instance, a blue edge indicates a significant interaction in the normal group. For more examples and views see Section 3. 2 Installation NAViGaTOR requires a minimum of 20 MB of free hard disk drive space, the Java Runtime Environment (JRE) 1.5 or higher, and OpenGL drivers. A system with at least 256 MB of RAM and a 1 GHz processor is also recommended. To install NAViGaTOR, go to and download a copy containing precompiled binaries appropriate for your operating system. Save and run the downloaded file to launch an installation wizard. 2

3 3 Getting Started To load a first data set, select the File/Load Text File menu item (Figure 2), causing a dialog box to appear. In this dialog box, go to the folder where NAViGaTOR was installed, select the docs subfolder, then select the sampledata subfolder, and then select a sample data file (e.g. tab delimited data.txt). Once a data file is selected, a 2nd dialog box appears where two groups must be selected. For example, double click on Control to make it the first group, and then double click on Experiment 1 to make it the second group, and click Ok. Then, a 3rd dialog box appears where the data may be filtered by Source. For example, click Select All to select all the sources, and then click Ok to finish. The loaded network should now be displayed in the main window. Figure 2: The File menu contains menu items to load and save data. 3.1 Force-Directed Layout NAViGaTOR uses force-directed layout to position the nodes of the network. Repulsive forces are simulated between non-interacting nodes, and spring forces are simulated between nodes connected by an edge. The simulation of these forces causes the nodes to gradually spread out and settle into a more-or-less stable layout. The user can influence this layout process by dragging nodes to different locations and by fixing their positions (double clicking on a node toggles whether it is fixed in place or not). The simulation of forces can also be paused via the Pause button in the control panel on the bottom of the main window. 3.2 File Formats NAViGaTOR supports loading and saving data in four formats: (1) Text File, a simple tab-delimited text file with metadata; (2) Network File (.nwk), a binary Java object file; (3) PSI-MI (Proteomics Standards Initiative s Molecular Interaction), which is an XML format; and (4) Navigator XML 1, an XML format developed at Jurisica Lab. Loading of data can also be done by issuing an online query to OPHID (Online Predicted Human Interaction Database, causing an interaction network to be dynamically constructed based on the results. 1 Referred to as Internal XML in the File menu; this will be changed to Navigator XML in the next release of the software. 3

4 During a NAViGaTOR session, various information associated with the network is generated, such as node positions, shapes, and annotations. To save this associated information when saving a network, users may save using the Network File or Navigator XML formats. The Network File format stores the maximum of this associated information, however, it achieves this by storing a binary dump of data structures stored in RAM at runtime, and thus is not compatible across different versions of NAViGaTOR or across different platforms. For this reason, use of the Navigator XML format is recommended for saving networks and their associated information. More information on the supported data formats is given in the following subsections Tab-Delimited Text File Tab-delimited files start with metadata describing the interaction data contained in the file. For instance, refer to the sample file tab delimited data.txt. The metadata is delimited by the # symbol, followed by a name tag (tags are case sensitive) and the column in which the data it refers to is found. The sample file shows the four required tags: NumOfCols, Source, Node1, and Node2. The tab delimited format can also include data such as node ontology, node description, expression level values, etc. This is followed by the actual interaction data. Interactions are stored as binary pairs with each row containing information on a single interaction (two proteins) Navigator XML Format The Navigator XML format is a slightly richer information source and, in addition to the information contained in tab delimited text file, has tags for determining id type (unigene, swissprot, etc.) and gene synonym. This is the recommended format for saving files since it retains layout information and is supported across future versions. For detailed information on this format, refer to the XML schema available at The sample file internal xml data.xml is a sample data file in this format PSI-MI Format NAViGaTOR supports the PSI-MI format for loading and saving protein interaction data. This format is also supported by a number of protein interaction databases including the OPHID database ( PSI-MI is an information rich format, and NAViGaTOR tools for querying PSI-MI documents are under development. Details on PSI-MI can be found at The sample file psi format is a sample data file in this format. 3.3 Ontology, Expression levels, and Interaction Strengths As mentioned earlier, NAViGaTOR colour codes information such as protein ontologies, expression level, and interaction strength. This information has to be included in the input file for NAViGaTOR to apply an appropriate colour scheme. For example, refer to sample files internal xml data.xml and tab delimited data.txt under the docs/sampledata folder. These files contain the data related to ontology (ontology description tag), node expression (node1 and node2 median tags for each group), and interaction strength (correlation and t test tags). The ontology categories are determined by concatenating the GO (Gene Ontology terms and comparing them against an internal dictionary. By clicking on 4

5 the Legend icon in the tool bar along the left edge of the main window, a description of the classification of the node colour can be seen. Furthermore, if the Get Ontology function (in the Ophid menu, see Section 4.1.7) is used, then the GO terms are displayed in the side description panel. The edge color scheme is based on showing interactions occurring in different groups or under different conditions. The input file does not impose a limit on the number of groups involving a single interaction (row in the data file), however, interactions involving only three groups are shown in any given network. For the purposes of this discussion, we will consider the tab delimited file format (tab delimited data.txt), however, the Navigator XML format also supports such data (see the XML schema file at Each interaction group has 3 columns associated with it in the input file. The first column Grp1Correlation gives the correlation value for the interaction. The 2nd column gives the number of samples used to determine the correlation, and the 3rd column gives a significance value (t test value) based on number of samples identified in the 2nd column. Currently, the edge color will show a significant interaction if the t test value is less than or equal to 1%. Below is a detailed description of the edge legend: Blue: Significant interaction in Group1. Red: Significant interaction in Group2. Black: Significant interaction in both groups (this is the default when no group information is present). Light Grey/Dotted: No significant interaction in either of the groups. Figures 3, 4, and 5 show the different views for edge and node colour schemes. Figure 3: The basic NAViGaTOR colour scheme, in which nodes are coloured according to ontology categories, and edges are coloured according to significance of interaction in the different groups. This figure also shows a rectangular labels filter in the centre (invoked through the right-mouse-button menu), that can be dragged around and that causes labels of the nodes within the filter to be displayed. This figure also shows the collapsible description panel on the right which describes the selected node, partners and GO (Gene Ontology) information. 4 User Interface Functionality This section describes the functions available in NAViGaTOR. First, functions within each of four top-level interface components (the menu along the top edge of the main window, the tool bar along the left edge, the control panel along the bottom edge, and the description panel on the right (Figure 1)) are given, and then functions associated with the mouse and/or keyboard are listed. 5

6 Figure 4: Similar to Figure 3, but gradient edge colour based on correlation values for the interactions. Figure 5: Gradient node colour based on expression values. 4.1 Application Menu File Menu This menu contains options for loading, saving, and transforming data files. Load Text File: loads a tab delimited text file as described in Section Load Network File: loads a NAViGaTOR network file (.nwk). Load XML: Contains sub-menu giving options to open Navigator XML format files and PSI-MI XML files. 6

7 Load from Ophid: Executes an OPHID query based on protein id s input by the user and graphs the resulting protein interaction network. Save as Text File: Saves network as a tab-delimited text file. Save Network: Saves network as a network file (.nwk). This format is dependent on the release version of NAViGaTOR and thus should only be used for networks that do not require long term storage. Save as XML: Contains sub-menu giving the option to save the network in Navigator XML or PSI format (exporting to PSI format results in loss of information such as protein highlighting, node expression values, and edge correlations. Such information is not part of the PSI-MI specification). Text to XML: Opens a wizard that allows user to load data that is stored in a tab delimited text file but does not match the input formats specified in Section 3.2. The wizard allows the user to identify the column position of elements required to construct an interaction network. Preferences: Allows you to set your preferences. User can change the default home directory. Also allows user to set a different default node colour. The user may also choose to visualize the nodes as flat nodes which are nodes without any border. This generally speeds up the rendering of the network. Exit: quits Select Menu This menu includes options for selecting individual proteins in the network and performing operations on them such as highlighting, fixing position, etc. Select All: Selects all proteins on the network. Individual proteins as well as groups of proteins can also be selected. In order to select a single protein, left mouse click on the center of the node. To select a group of proteins, hold down the Alt button and the left-click button and drag the mouse over the proteins- releasing the button will highlight the proteins. Select None: Deselects current selected proteins on the network. De-selecting is done by a right mouse click on the highlighted node. Alternatively, to de-select a group of proteins, hold down the Alt button and the right-click button and drag the mouse over the proteins releasing the button will de-select the proteins. Invert Selection: Switches selection to proteins not currently selected. Highlighting: Contains a sub-menu which clears highlights (halo around the nodes). Additionally, contains a sub-menu for highlighting proteins on the network matching proteins listed in a separate text file. This function is useful when a user needs to highlight the original set of proteins which were used to build a complex network incrementally through OPHID searches. Select Highlighted: Selects highlighted proteins in the network. Expand Selection: Expands the selection to neighbors of the currently selected protein. Fix Selected: Fixes the position of selected nodes. 7

8 Unfix Selected: Unfixes the position of selected nodes. Export Selected Nodes: Exports basic information on selected nodes to a user selected text file View Menu Contains options for altering visual aspects of the network. This includes imposing different node/edge colour schemes, changing node sizes, changing node shapes, etc. Ontology Colouring: The default colouring option. Colours proteins according to ontology information (if available), and edges according to interaction significance based on t test values (if available). Gradient Colouring: Colours the proteins based on expression values. Edge Correlations: Colours the edges based on actual correlation values. Flip Groups: Swaps the groups, i.e. flips their ordering. For example, if Group 1 is normal and Group 2 is cancer, this function would set Group 1 to be cancer and Group 2 to be normal and change the edge colouring accordingly. Change Groups: Allows loading of different groups. Edge Labels: Labels the edges with the source information (1st column in tab delimited text files). Label: A menu item for node and edge labeling. It is composed of sub-menu items described below. Label All: Labels all the proteins on the graph. The label is either the first word in the description field or protein id. Label All (2D): Does the same as above, however, the nodes are given 2D shapes (an ellipse is the default shape). See Shape Selected option below. Label None: Removes labels from all nodes. Label Selected: Labels the currently selected protein. Label Selected (2D): Labels the currently selected protein and activates the default 2D shape (ellipse). Toggle Selected: Toggles the currently labeled nodes (inverts the selection and labeling). Label Neighbours: Labels the neighbours of the currently selected proteins. Node Size: Changes the size of the nodes. Shape Selection: Changes node shapes. Add Layer: Option for expanding (adding interactions) to the currently selected protein. This options opens the same dialogs as when loading a new interaction network. However, in this case, only those interactions involving the currently selected node are added (if not already present). 8

9 Remove Layer: Removes the currently selected protein and all proteins it interacts with Export Menu Export BMP: Exports current network layout to a.bmp image file. Export JPEG: Exports current network layout to a.jpeg image file. Export Pajek (.net): Exports current network to a.net network file readable by Pajek Export PDF: Exports current network layout to a.pdf file. Export SVG: Exports current network to SVG format. Export TIFF: Exports current network layout to a.tif image file Layout Menu FR random: A grid variant force-directed algorithm which randomly applies the repulsive force to 1/3 of the nodes. Less processor-time intensive therefore it works well with large networks. FR layout algorithm: Applies repulsive force to all of the nodes. This is a more computationally expensive algorithm; however, a stable configuration can be obtained quickly for smaller networks Analysis Menu Contains tools for analyzing and comparing interaction networks. Compute Cliques: Computes components of the network that are highly interconnected. Technically, a clique is a complete subgraph, i.e., a subset of nodes where each node has an edge to every other node. For efficiency purposes, this command will only compute approximate cliques, i.e. subgraphs that are approximately complete. Compare Networks: Gives an option of selecting two networks and creates a union of the networks (in terms of nodes) and also does pair-wise comparison of the edges if different groups are used. See tutorial for comparing networks on the NAViGaTOR website. Interaction List: Prints out on the standard output (the command line), the list of all interactions in the network. Highlight Hubs: Identifies the top percentage of nodes with the greatest number of edges, i.e. the greatest number of interactions, where the percentage is supplied by the user. These nodes with the greatest number of edges, called hubs, are then highlighted. Collapse Hubs: Identifies the top percentage of hub nodes, as above, and collapses them into composite nodes. 9

10 4.1.7 Ophid Menu This menu contains options for connecting to OPHID database. A key functionality is to expand on existing networks by submitting id s of proteins on an existing panel to OPHID. This gives a layered approach to building a complex network. For all OPHID related features, a live internet connection is necessary. Get Ontology/Description for All Nodes: Retrieves ontology information (GO (Gene Ontology) categories and terms) and descriptions for all nodes on the network based on the user selected id. Search Ophid for Selected Nodes: The id s of selected nodes are submitted to OPHID and the resulting interactions are added to the existing network D Options Menu Enable 3D View: Enable 3D node layout Cylindrical Edges: Render edges as cylinders (computationally expensive) 4.2 Application Tool Bar The tool bar is placed on the left of the graph panel and contains command options listed below. Collapse Nodes: Collapses the selected nodes into a single composite nodes. The interactions of the composite node is based on the average value of the interactions of its components (if edge correlation values are present). Expand Nodes: Recovers the original nodes and interaction from a collapsed/composite node. Delete Highlighted Nodes: Deletes a node and all neighbors which do not have any neighbors other than the node being deleted. Toggle Legend: Displays/removes legend for node and edge colours. Switch Selection Mode: Allows the user to switch between graph or node selection mode in the 3D view. Copy Nodes: Copies selected nodes and their neighbourhood to a new graph panel. 4.3 Application Control Panel This is the control panel at the bottom of the graph panel and it contains commands for the force directed algorithm and variables related to neighborhood functions. Steps: The number of steps used to generate neighborhood function (see local neighbourhood function under Section 4.5). 10

11 Force: This is a variable controlling the repulsive force between nodes. Increasing this value forces the nodes to disperse further apart. Protein Search: A text box to search for nodes on the network. The search term could be the node id, node label, any string from the description or GO information. If the search is successful, the node is highlighted. For example, you can search for all proteins which contain the word matrix in their description or with a particular GO Id. Clear: Disposes the current graph panel. Pause: Stops the force-directed algorithm temporarily. This stops the movement of the nodes. Fix: Fixes the position of all nodes on the graph panel (but does not stop the force-directed algorithm). This means a user can unfix some nodes (by double clicking on them) to let the force-directed algorithm reposition them. 4.4 Description Panel This is the panel on the right hand side of the graph panel and it contains tabs to display information about the node and the network. Network tab: Allows the user to write comments about the network. It will be saved by either clicking on the save button or hitting enter. In order to view and store the comments permanently with the network, the user must also save the network in the Navigator XML file format. Node tab: Displays the name of the gene, which by default is the first word of the gene description. This gene name can be changed by the user and is saved by clicking the save button or hitting enter. The display panel also shows a list of the partners of the selected node. Also, if GO information is available for the selected node, it will be displayed on this display panel. 4.5 Right-Click Menu This menu is activated by right clicking the mouse on the network. Randomization: A right-click menu item for randomizing the network. It is composed of sub-menu items described below. Randomize All: This randomizes the position of all the nodes in the network. Randomize Unselected Nodes Only: This randomizes the position of all the nodes which haven t been selected. Users may fix the selected nodes to prevent them from being affected by the changing forces during randomization. Visualization: A right-click menu item for changing the visualization of the network. It is composed of sub-menu items described below. Highlight Colour: Allows the user to change the highlight colour of subsequent highlighted nodes. 11

12 Blend: Opens a Blend Controller which allows the user to change the contrast of the unselected nodes so that the selected nodes are displayed more prominently. The user can also change the amount of blending of the selected and non selected edges. Filters: Displays a rectangular, draggable window or filter through which either node labels or interaction sources can be read. If the right-click button is used to click the center of a node then an additional menu item for node related features is displayed: Node Related: A right-click menu item for viewing different items relating to the selected node. It is composed of a sub-menu items described below. Local Neighbourhood: Opens a new graph panel and populates it with the selected node and all its neighbors which are a fixed number of edges (steps) away from it. The number of steps can be selected from the control panel at the bottom of the graph panel (see steps chooser in the control panel). Complex: This function is similar to local neighborhood function, but in this case, all nodes reachable from the selected node are included in the new panel. Reachable refers to nodes which can be reached via a path from the selected node. Description: Opens a dialog giving a full description of the selected node. This view is also available via a collapsible description panel on the right hand side of the main panel (see description panel in Section 4.4). 12

13 4.6 Mouse and Keyboard Controls This section outlines some mouse and keyboard shortcuts available to the user. In the below, Left and Right refer to the left and right mouse buttons, respectively. Left-click to select a node. Left-drag to move a node. Alt-Left-drag to rectangle-select a group of nodes: While holding down the Alt key, groups of nodes can be selected by dragging and holding the left mouse button over the nodes. Once the button is released, the nodes will be selected. Alt-Right-drag to rectangle-deselect a group of nodes: While holding down the Alt key, groups of nodes can be unselected by dragging and holding the right mouse button over the nodes. Once the button is released, the nodes will be unselected. Ctrl-Left-drag to move all selected nodes: While holding down the Ctrl key, all of the selected nodes can be pulled to a different place on the graph panel by holding the left click button down and moving the mouse. Once the button is released, the nodes will move to their new location. If the selected nodes have been fixed, they will move together but retain their relative position. Ctrl-Right-click to deselect a node: While holding down the Ctrl key, a single selected node can be unselected by clicking on the right mouse button. Once the button is released, the node will be unselected. Left-double-click to fix/unfix the position of a node: Double clicking on the centre of a node fixes the selected node in place. Double clicking on a fixed node releases it. Ctrl-A to select all. Ctrl-D to deselect all. Ctrl-I to invert the selection. 5 Additional Resources 5.1 Tutorials Tutorials for using NAViGaTOR are available on the NAViGaTOR website at This includes an introductory tutorial on how to load a protein-protein interaction network and using the basic features. There are also advanced tutorials for learning about the tools available to analyze the proteinprotein interaction network. 13