User`s Manual
Contents
1. 0 0 thd a M A with Dan MOdelling User s Manual Contents 1 Requirements and installation 1 1 Java 1 2 Cytoscape 1 3 UPPAAL 1 4 Installing ANIMO 2 ANIMO user s manual 2 1 Modelling a small network 2 1 1 Managing simulation data and activity levels plots 2 2 Additional tips SA eg ck OY 2 2 1 Editing a network in Cytoscape 2 e 2 2 2 ANIMO features da ed ee ee a a e Eh Eo 22d A o A II 224 Update ANIMO ot owe e E eee e A e EES 10 O 0 o oo Q A F amp F N N N e e 1 Requirements and installation In order to run ANIMO a desktop or laptop computer is needed with the following software installed e Java see Section 1 1 e Cytoscape www cytoscape org see Section 1 2 e UPPAAL www uppaal org see Section 1 3 The software to run Java based programs is provided for free by Oracle More information is avail able on the java com website Cytoscape is an open source project released under the terms of the GNU Lesser General Public License UPPAAL is developed by a collaboration by the universities of Uppsala Sweden and Aalborg Denmark and is free for non commercial applications in academia only All of these softwares work under Windows AY Mac OS and all most common GNU Linux A distributions If the requirements are already met ANIMO can be directly installed following the instructions in Section 1 4 Note when required to type something the text to input will be rep2. ote O USO O Ol oe O Gedo ee O on DO O E A f gt _ _ __ _ _ aseun O uu awIL 08 09 Op oz noo i OL OZ Of OV OS 09 OL 08 A asoj e ep uoepnws BABS 3 13 abueyo AWAIpY puaba7 n e Aanoe ulajold 110M 3U azeuy dejs spuodas T 3o d ejlan UOIJEIASp p3s pue abeJan sun OT amndwo f seynuiw ozt hun uonenws synsey OWINY is jaued syns y ao A10M19N a H Bue d o1quo 5 e eE djeH sulbnjd ynoke7 Pajas m l PPI lld u0 ssas MAN dozy s a adexsoVAD 3 The completed example where also the feature that allows to view the activity levels of reactants at chosen simulation times is demonstrated the vertical red bar in the graph on the right can be moved through the slider under the graph and indicates the point in the time series on which the colouring of the nodes in the Network window is based The legends for colours and shapes can be found in the Legend panel Figure S3 zoomed can then be selected by dragging a rectangular selection around it see the definition of rectangular selection on page 8 e Zoom extents bring the zoom level back to default cancelling the effects of any Zoom rectangle command Whenever the result of one or more simulations is shown as a graph it is possible to use the slider under the graph to move through the entire simulation showing the activity levels of all reactants represe
3. verify Timed Automata models You can find it in the bin bin Linuzx bin Win32 depending on your operating system directory inside the UPPAAL installation directory or where it was copied at step 5 for ES in Section 1 3 Click the Close button to close the Manage Plugins window ANIMO is correctly installed and ready to be used 2 ANIMO user s manual We will now present a step by step sequence to obtain an example model with ANIMO which will allow us to illustrate the main features of the tool 2 1 1 Ze Modelling a small network Run Cytoscape If Cytoscape is already running and there are open documents please make sure that the current work is saved before proceeding From the File menu select New gt Session Answer positively to the question Current session all networks attributes will be lost Do you want to continue From the File menu select New gt Network Empty Network In the Control Panel find the Editor tab If you cannot find it click the black arrows on the top right of the panel to search through the available tabs Click the name of the tab to activate it Add 5 nodes to the empty network by Ctrl clicking on empty areas of the Network window Note Ctrl clicks are obtained as follows While holding the Ctrl key down click with the left mouse button then release the Ctrl key The Ctrl key is usually located in the lower left or lower right corner of the ke
4. Edit reaction item from the menu Use that window to set the parameters of the edges as indicated in Table S2 The settings for the edge A gt B should reflect the ones shown in the Edit reaction window in Figure S2 Note In order to insert a qualitative parameter like the ones required by the example network click once the slider in the parameter box to activate it and then move the slider to match the requested value Table S2 The settings for the edges interactions in the example Interaction Influence Scenario Parameter value A gt 8B Activation 1 Medium BoC Activation 1 Slow C gt B Inhibition 1 Fast B gt D Activation 1 Slow E gt D Inhibition 2 V Slow 10 In the Control Panel activate the ANIMO tab by clicking its title 11 12 13 Click the Choose seconds step button A new dialogue window will appear you can safely choose a time resolution of 1 second per step and click OK Click the Analyse network button After a few seconds the Results Panel should appear on the right showing a plot of the activity level of reactant D over a time course of 120 minutes Figure S3 shows the resulting network and graph plot e Reaction A gt B Influence 8 Activation O Inhibition Reaction kinetics Scenario 1 k E E active A l l w slow slow medium fast v fast Save Cancel Figure S2 The Edit reaction window modifying the properties of edge A gt B 2 1 1 Man
5. aging simulation data and activity levels plots Each time a simulation result is obtained a new tab is added to the Results Panel see the right part of Fig S3 in which we identify three buttons a plot of the activity levels of the selected reactants and a time slider Clicking on the button Change title allows to select a new title for the tab this can be useful e g when comparing different simulations made on similar configurations of the same network Button Save simulation data allows to save the simulation data of the current tab on a sim file which can then be loaded and inspected in the future The Load simulation data button in the Control Panel above the Simulation box can be used for this purpose Please note that the best results are obtained only when loading a sim file when the Network window contains the same network on which the simulation data are based If no network is currently opened a network will not be opened by loading a sim file The Close button is used to close the currently displayed results tab Right clicking inside the graph area will bring up a menu that allows to perform some basic operations with the graph and its data e Add data from CSV superpose the graph with other data series found in a csv comma separated values file This file type can be obtained for example by exporting data from the default Excel format If you want the data in the csv file to be rescaled so that its maximum Y valu
6. e coincides with the maximum in the plot the data file needs to contain a column named exactly Number_of levels on the first row of which the maximum of the scale for the csv data needs to be put For example if the data in the csv file are on a 0 100 scale the value for Number_of_levels will be 100 e Save as PNG save the graph as it is shown in a png image file This file format can be opened by most image editors e Export visible as CSV export to a csv file all the series that are currently visible i e not hidden in the graph e Clear Data clear the contents of the graph removing all series This can be useful for plotting a csv file without superposing it to the current graph or for loading a file in which all hidden data where removed exporting the visible graph to a csv with the previous command e Graph interval change the lower and upper bounds for X and Y axes e Zoom rectangle zoom the graph around a user chosen rectangular area After selecting this command the shape of the mouse cursor changes into a cross The area of the plot to be NYd 0 ep 421 2 3 ppiyy WOOZ 07 Beup 4919 3461 Egz adessoyi 5 0 SUIOIjB MM J35M018 SINQGUY 410MJ3N 13SMOJ1g 3INQUIIJY abp3 J35M018 3INQ113IY 3PpON A10623 8 gt ulaj old JOE uoduosueJL O J03da33y O a _ dl a Oy E A AE fag asejeydsoyd H jeued ea OZT 80T 96 Y8 ZZ 09 8b de vz ZI 0 seo E O ovo omo O oO oon pro ooo ooo oo O O ooo wo
7. hading both bars and shading bars and symbols none by right clicking the corresponding line in the legend Symbols associated to a representation of standard deviation can be changed by Shift right clicking holding down the Shift key right click on the corresponding line in the legend Standard deviation values can be obtained when asking for multiple simulations in the network analysis but they can also be present in a csv file e g when the file contains averages of experimental data In a csv file the column containing the standard deviation values for column A should be named A_StdDev for the program to recognize it and properly display the data series with the associated error values 2 2 3 Parameter settings The application of some basic strategies when setting the parameters for a network allows the less experienced users to considerably shorten the modelling time First of all it is important to proceed in a top to bottom order trying to match a component to the corresponding data before inserting the components downstream thereof Second when choosing the kinetic parameter for an interaction we advise to first use the qualitative settings very slow slow medium fast very fast this allows to define the relative speeds of the interactions as soon as possible leaving the more precise parameter setting procedure as a follow up step Finally as can be seen from the parameter settings of Section 2 1 in
8. ions message is shown Java has been successfully installed Otherwise please try again or contact Java support Go to Applications Utilities Java Preferences If the Java Preferences window is shown Java is already installed otherwise the system will prompt you to install it Follow the instructions and Java will be correctly installed at the end of the procedure A If you run Ubuntu open the Software centre search for java and select OpenJDK Java 6 Runtime An Install button will appear next to the name of the package click that button and Java will be correctly installed If you run another distribution use your package manager in a similar way If you cannot find OpenJDK there may be the possibility to install Oracle Java Development Kit JDK instead 1 2 Cytoscape Cytoscape can be found at the address www cytoscape org download php an automatic installer program can be downloaded Please note that you need to register and accept Cytoscape s terms of use before being able to start the download Choose the latest 2 x version 2 8 3 on the left column possibly using a platform specific installer For Windows you can choose 64bit only if you know that your computer can run 64bit programs otherwise it is safe to choose 32bit Please note that Cytoscape version 3 is currently not supported 1 3 1 UPPAAL Point your browser to www uppaal org Note UPPAAL is free only for academic use Informati
9. nted with different node colouring in the Network window on the left For an example see Figure S3 the vertical red line in the graph represents the time instant on which the colours of the nodes in the Network window are based and can be moved with the slider over which the mouse cursor is drawn 2 2 Additional tips 2 2 1 Editing a network in Cytoscape Nodes and arcs can be placed in the network as shown previously with the Editor tab selected in the Control Panel Ctrl click click while holding the Ctrl or 36 key in an empty place to add a node Ctrl click the source node and click the target node to add an arc It is also possible to drag and drop the node arc icons from the Control Panel into the Network window Note to perform drag and drop move the mouse cursor over the icon in the Control Panel click with the left mouse button and without releasing the button drag the mouse cursor on the Network window where you want to place the symbol then release the left mouse button In order to delete a node edge select it by clicking or grouping them in a larger rectangular selection and then press the Delete key on the keyboard or select the Edit Delete Selected Nodes and Edges menu command Note in order to obtain a rectangular selection left click in the Network window where the upper left corner of the rectangle should be and without releasing the left mouse button drag the mouse cursor to where the lower right corner of the
10. on and contacts for commercial licenses can be found on the web site Click the Download link and choose the latest development version at least 4 1 for your operating system Fill in the required contact information and click the Accept and download button to download UPPAAL Note problems with the registration on UPPAAL website have been reported when using some versions of Microsoft Internet Explorer If the registration is unsuccessful please consider updating Internet Explorer or changing your web browser Unzip the downloaded file to a known location UPPAAL will be installed there Complete UPPAAL installation ES Open the UPPAAL installation location in Finder drop the UPPAAL App icon in your Applications folder and copy the verifyta executable file to a known location The installation of UPPAAL is complete go to Section 1 4 Au A Open a console this was done in Sec 1 1 step number 1 type cd PATH_TO_THE_ UPPAAL_DIRECTORY and press Return PATH_TO_THE_UPPAAL_DIRECTORY is the path to the directory where you installed UPPAAL It can be for example c Users myuser Desktop uppaal 4 1 15 or home myuser programs uppaal 4 1 15 An Note some Windows users may have access only to specific partitions D Z in that case please first change to the corresponding drive letter where the downloaded file was extracted For example if UPPAAL is located in d myuser Programs uppaal 4 1 15
11. onding arrow and a new window will open allowing you to choose a new colour clicking OK will accept the new colour The modifications made in the Gradient Editor for Node Colour window should be automatically reflected in the model when the gradient is as wanted simply close the window If the node colours seem not to have been updated please move the slider under an existing graph in the Results Panel 2 2 2 ANIMO features Nodes and edges and groups of nodes edges highlighted via a rectangular selection can be disabled by choosing ANIMO Enable disable from the right click menu they will be represented with less e Gradient Editor for Node Color _ Continuous Mapping for Node Color dv y v gt 0 0000 0 5000 1 0000 Min 0 0 activityRatio Max 1 0 Range Setting 1 ES Min Max Delete Figure S4 Changing the colours used to represent node activity in an ANIMO model saturated colours and can be re enabled by performing the same action Moreover a node can be enabled disabled directly in its properties window where it is also possible to add remove the node from the list of series appearing in the graph resulting from a simulation of the network by selecting Plotted or Hidden see also Figure S1 nodes that will be plotted are circled in blue Every enabled node will be taken into account when computing the evolution of the system but only nodes marked as Plotted will appear in a graph Each plot in an ANIMO Re
12. order to obtain a peak behaviour it is particularly important that a negative feedback is present as an example see the interactions involving B and C in Tab S2 and that the inactivating interaction in the loop is faster than the ones activating the target node A final note on the seconds step button This button allows to define the time granularity of the simulations but it is not strictly necessary to choose a very precise value If the current value for seconds step is too high or too low to allow the network to be properly simulated ANIMO will automatically choose respectively the highest lowest value that still allows to avoid rounding problems It will be possible to notice such a change in the value of time scale when the number on the seconds step button changes 2 2 4 Updating ANIMO To check whether a new version of ANIMO has been published run Cytoscape and ask for an update of all plug ins via the menu command Plugins gt Update Plugins After some seconds during which Cytoscape will contact all the providers of the installed plug ins the system should report the list of updatable plug ins Note a window with the message Attempting to connect to XYZ may appear and disappear multiple times it is the normal behaviour If an updated version of ANIMO is available it will appear under the category Updatable Plugins Analysis ANIMO v1 38 If no plug in can be updated a message stating No updates available for cu
13. rectangular selection should be then release the left mouse button All the entities which were even partially touched by the rectangle are now selected Navigation inside the Network window can be performed by clicking and dragging the centre mouse button while zooming can be done by either rotating the mouse wheel or clicking and holding the right mouse button while moving the mouse in a vertical direction Finally note that the colours used to represent node activity can be changed using the VizMapper interface provided by Cytoscape changing the setting for Node colour shown in Figure 4 To change the node colours activate the VizMapper Y tab in the Control Panel and find the entry named Node Colour in the Visual Mapping Browser box Click the arrow shaped icon directly to the left of Node Colour the current setting for the node colours should appear click the coloured bar to open the window shown in Figure 4 The activityRatio on which the node colouring is based is the ratio between the current activity level of a node and its number of activity levels The coloured arrows pointing downward on the upper border of the coloured rectangle can be dragged along the length of the 0 1 interval thus changing the point at which a particular colour appears New arrows can be added with the Add button while clicking an arrow and pressing the Delete button will remove one To change the colour of a point in the interval double click the corresp
14. resented like this the quotation marks are not intended to be typed In case of problems accessing a web site with Microsoft Internet Explorer we advise to try with a different web browser such as Mozilla Firefox or Google Chrome or to update Internet Explorer 1 1 Java 1 In order to check that Java is installed open a console En Windows 7 press Windows button and type cmd then press Return Previous versions in the Start menu find All programs gt Accessories Command Prompt Go to Applications gt Utilities gt Terminal A Under Gnome press Alt F2 type gnome terminal then press Return Under KDE press the KMenu button type konsole and click Konsole Under Unity press the home button the one with the Ubuntu logo 3 type terminal and click the Terminal icon 2 Type java and press Return If a brief error message like unknown command is shown Java needs to be installed please proceed to step 3 Otherwise please continue to Section 1 2 3 An Point your web browser to java com Click Free Java Download then on Agree and Start Free Download If asked for permission to run the installer grant that permission After the download double click the installer and install Java following the guided steps To check that Java has been correctly installed a web page is automatically opened at the end of the installation process Click on Verify Java version If a Congratulat
15. rrently installed plug ins will be shown 10
16. sults tab contains by default a legend which can be used to modify which series are displayed and how they are displayed Clicking with the central mouse button on a series name will hide it from the graph while the same centre click the coloured line beside the series name will change the colour of that series cycling through a predefined set of available colours The entire legend can be hidden by clicking with the central mouse button anywhere on the eraph not inside the legend or it can be dragged around by clicking and holding the left mouse button releasing it when the preferred position is reached Rotating the mouse wheel will allow the thickness of all the graph lines and the size of the text to grow or decrease this feature can be useful when the window containing the graph is very large As the model is non deterministic i e its evolution will not be exactly the same for every single simulation run it is possible to ask ANIMO to perform a number of simulation runs in a batch plotting the averages of the activity levels over the runs together with a standard deviation value or showing a so called overlay plot where all runs are plotted over each other The controls that allow to ask for multiple simulation runs can be found in the Control Panel inside the Simulation box Standard deviation may be represented in the graph it is normally shown as vertical bars but its aspect can be cycled through five possibilities vertical bars s
17. the two commands to be entered are d 9 cd user Programs uppaal 4 1 15 Type java jar uppaal jar and press Return The license for UPPAAL will be automatically acquired and the main window of UPPAAL user interface will appear you may now close that window Installing ANIMO 1 ANIMO is free only for academic use For commercial licenses please contact us Run Cytoscape Click the menu command Plugins Manage Plugins the Manage Plugins window will open Select the Settings tab and press the Add button 10 11 Insert this Name ANIMO and this URL http fmt cs utwente nl tools animo plugins xml please note the http is required then confirm with OK A development less stable version of ANIMO is also available to try it use the URL http fmt cs utwente nl tools animo dev plugins xm1 instead of the one given above From the Download Sites list in the upper part of the Manage Plugins window select ANIMO you may need to scroll down it should appear after Cytoscape The panel on the left shows a smaller list of plugins under Available for Install gt Analysis select ANIMO v1 38 if you chose for the development version of ANIMO the number will be 2 x Click the Install button The ANIMO tool will be automatically downloaded and installed The tool will ask you to indicate the position of the verifyta executable which is the tool to
18. yboard Apple keyboards may have the symbol instead of Ctrl The Edit reactant dialogue window is opened when a new node is added or when you right click an existing node and then select the ANIMO Edit reactant item from the menu Use that window to set the properties of the nodes as indicated in Table S1 taking the setting in Figure S1 for node A as reference When the properties of a node have been inserted confirm the choice with the Save button Table S1 The settings for the nodes signalling network components in the example Total act Initial act Name levels level Molecule type Enabled Plotted A 15 15 Cytokine Yes No B 15 0 Receptor Yes No C 15 0 Other Yes No D 100 0 Kinase Yes Yes E 1 1 Phosphatase Yes No 8 In order to add edges to the network make sure that the Editor tab is still active in the Control Panel and add the following edges by Ctrl clicking the source and then clicking the target A gt B B gt C C B B gt D E gt D e Edit reactant Total activity levels 15 ASS ld las hs ji a ql BL ML 100 Initial activity level 15 Molecule type Cytokine I I AL Ly al ot I O MIA da e Enabled _ Disabled Plotted Hidden Cancel Figure S1 The Edit reactant window modifying the properties of node A 9 The Edit reaction dialogue window is opened when you add a new edge or when you right click an existing edge and then select the ANIMO
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