User's Manual - Formal Methods and Tools
Contents
1. 0 0 thd a om with Dan MOdelling User s Manual Contents Requirements and installation 1 1 Java 1 2 Cytoscape 1 3 UPPAAL 1 4 Installing ANIMO ANIMO user s manual 2 1 Modelling a small network 2 1 1 Managing simulation data and activity levels plots 2 1 2 Editing a network in Cytoscape 2 2 ANIMO features 2 3 Parameter settings 2 4 Opening a Timed Automata model generated by ANIMO 2 5 Updating ANIMO 2 6 ANIMO Options OQ A B DB WH WN KF e PrP ae oO 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 software work under Windows 48 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 represente2. message is shown Java has been successfully installed Otherwise please try again or contact Java support A If you run Ubuntu open the Software centre search for java and select OpenJDK Java 7 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 In any case please make sure that the installed Java version is at least 7 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 version at least 3 2 0 possibly using a platform specific installer You can choose 64bit only if you know that your computer can run 64bit programs otherwise it is safe to choose 32bit 1 3 1 UPPAAL Point your browser to www uppaal org Note UPPAAL is free only for academic use Information 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 NOT 4 0 for your operating system Fill in the required contact information and click the Accept and download button to dow
3. made on similar configurations of the same network Reset to here restore the network to the state it was in when the simulation that gave the current graph was performed If no simulation was done with the network in its current state the current network configuration will be lost Difference with allows to compute a difference between two simulations After pressing this button select another graph i e another tab in the Results panel and click on the Difference with this button a new graph will be shown highlighting the differences between the two selected graphs first second The slider under the graph can be used to visualize the differences in node activity over the course of the simulation using this color scale red the node is less active in the first simulation than in the second green more active white similar activity level 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 Close close the currently displayed graph Right clicking ins
4. ones available All changes performed in this dialogue are immediately reflected in the graph e Zoom this sub menu contains commands to change the scale of the graph managing its zoom level Graph interval change the lower and upper bounds for X and Y axes 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 zoomed can then be selected by dragging a rectangular selection around it see the definition of rectangular selection on page 9 Zoom extents bring the zoom level back to default cancelling the effects of any Zoom rectangle command e Graph type this sub menu allows to change the type of the graph using different ways to plot the data Step shaped graph in order to display more faithfully data series with low granularity this option can be chosen It explicitly keeps each data series constant until the next point in which its value changes thus producing a graph that contains many steps Normal graph the default way to display data series from a simulation plot the activities as lines using the X axis to represent simulation time and the Y axis to represent activity levels Heat map graph alternative way to represent activity levels use the activity scale as defined in the Legend panel the colours can be changed using Cytoscape s visual style options for node
5. should start select Add Edge and click on the node where the edge should arrive Add the following edges A gt B B gt C C gt B B gt D E gt D The Edit interaction dialogue window is opened when you add a new edge or when you right click an existing edge and then select the ANIMO Edit interaction item from the menu or when you double click on an existing edge Use that window to set the parameters of the Edit reactant Total activity levels 15 Name A ie Ve lee ee ee 1 21 41 61 81 100 Molecule type Initial activity level 15 Cytokine v EEEE 0 3 6 9 ab Ls Enabled Disabled Plotted Hidden Description Cancel Figure 1 The Edit reactant window modifying the properties of node A The Description box in the Edit reactant dialogue can be used to add comments about what a node represents or where its reference was found in literature when keeping the mouse cursor over that node in the network the description text will be shown as tool tip 10 11 12 13 edges as indicated in Table 2 The settings for the edge A B should reflect the ones shown in the Interaction A B window in Figure 2 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 2 The settings for the edges interactions in the examp
6. 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 Export this sub menu contains options to export the currently visualized data 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 Export visible as CSV export to a csv file all the series that are currently visible i e not hidden in the graph e Manage data series opens a new dialogue window see Fig 4 that allows to manage the series plotted in the graph mostly with the same effect as the shortcuts explained in Subsection 2 2 The available features are manually sort the data series they are sorted alphabetically by default by dragging and dropping them using the handles on the left choose whether to show or hide a data series by checking or unchecking the corresponding checkbox rename a series using the text box change the colour of a series choosing among the list of available colours change the way a series shows its error bars when supported using one of the available methods in case a series with error bars is to be displayed using dots option Dots and bars for the error bar type choose the shape of the dots among the
7. and select Add Node to add a node right click on the source node and select Add Edge then click on the target node to create a new edge between the two nodes In order to delete a node edge select them 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 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 In case no central mouse button is available the Network tab in the Control Panel will allow to navigate the network together with the zoom buttons available in Cytoscape s toolbar where the open and save icons are Finally note that the colours used to represent node activity can be changed using the Style tab in the Control Panel Please refer to Cytoscape s manual to do that 2 2 ANIMO features N
8. colour to plot the activity levels against simulation time Each data series occupies a fixed position on the Y axis while the X axis is used to represent time When 10 or more series are chosen to be plotted this option is automatically selected X vs Y plot the values of one data series against another A dialogue will open that allows to choose among the series currently present in the graph which one should be represent by X axis and which by Y axis When in this mode the slider under the graph will move a dot on the graph instead of a vertical bar as different time points have different coordinates both in X and Y 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 Manage graph series Title Color E e fa E Title _ Color pa C B Title _ Color nia IG C D Change plotted series s Close Figure 4 The Manage graph series dialogue reorder hide show rename change the colour and error bar style of the series present in the graph The option to change the way error bars are represented for a data series is present only if that data series has an associated standard deviation This situation occurs when the data series represents the average of a number of simulations see the de
9. d 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 Ay Windows 7 press Windows button and type cmd then press Return Previous versions in the Start menu find All programs 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 Au amp 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 Congratulations
10. er to rescale a set of reactions select the corresponding edges in the Network window right click on one of the selected edges and choose ANIMO Rescale k value s from the menu In the new dialogue insert the scale factor that will be applied to the selected reactions and press OK the new values will be automatically computed and set by ANIMO 2 4 Opening a Timed Automata model generated by ANIMO All Timed Automata models generated by ANIMO in a Cytoscape session are available until Cy toscape is closed and can be usually found in the system temporary directory In order to know exactly where the Timed Automata model for a particular simulation was saved check the Cy toscape log small button on the lower left most corner in Cytoscape it contains the advancement status of all the executed tasks including ANIMO simulations Before beginning the analysis each simulation task explicitly outputs the location of the file that is going to be analysed Opening the same file with UPPAAL will allow the interested user to inspect the model by themselves and query it for other properties directly in the UPPAAL user interface 2 5 Updating ANIMO To check whether a new version of ANIMO has been published run Cytoscape and open the app manager via the menu command Apps App Manager In the Check for Updates tab the available updates will be listed if a new version of ANIMO was published it will appear in the table Select it and p
11. ide 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 No oway ull wiL 08 09 OV OZ ne AAPL uad soj ezep uonejnws BABS UUM BIUSIBYIG Sis O 7 S Y V auey syns y OWINY xo Jeued sj nsey 0 WAOMION suondo 13430 O Awwng o vnuw Y u 9 V Joye uonduosuel O asejeydsoud seuy O 10 d 2 4 O auo Asobaje9 ul 401d ul AYNPY pu q Bup y Jomz au apo azsjeuy Jay Jeyewesed jojd Aeyang UOIJEIASp pys pue a elany suna oTt ayndwog f 7 seynuiw OzT un uonenwIs ejep uonepnws peol uoIss Ss M N UOISS S Figure 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 using 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 default Excel format If you want the data in the csv file to be rescaled so that its maximum Y value coincides with
12. ient and is se lected by default The difference between the two models can be seen by opening the models generated by ANIMO when each choice is made 11
13. itively to the question Current session all networks attributes will be lost Do you want to continue From the File menu select New Network Empty Network Click OK in the Create New Network dialogue A new white window will open there we will insert the nodes for the new network Add 5 nodes to the empty network by right clicking on empty areas of the Network window and selecting Add Node Note right clicks are available only on mice with at least two buttons On Apple mice or devices with only one button it is necessary to keep the button with the 8 symbol or possibly Ctrl pressed while clicking The Edit reactant dialogue window is opened every time a new node is added or when you right click an existing node and then select the ANIMO Edit reactant item from the menu or when you double click on an existing node Use that window to set the properties of the nodes as indicated in Table 1 taking the setting in Figure 1 for node A as reference When the properties of a node have been inserted confirm the choice with the Save button Table 1 The settings for the nodes signalling network components in the example Total act Initial act Name PO leval 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 In order to add an edge to the network right click on the node from which the edge
14. le Interaction Influence Scenario Parameter value A gt B Activation 1 Medium BoC Activation 1 Slow C gt B Inhibition 1 Fast BD Activation 1 Slow E D Inhibition 2 V Slow In the Control Panel make sure that the ANIMO tab is selected by clicking its title Insert a value of 120 minutes in the Simulation box Click the Analyse network button alternatively you can press Return after inserting the number of minutes 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 3 shows the resulting network and graph plot Interaction A gt B Influence Activation Inhibition Reaction kinetics Scenario 1 k E E active A I l I v slow slow medium fast v fast Description Figure 2 The Edit interaction window modifying the properties of edge A B There is a Description box also for interactions it can be used with the same aim as for nodes 2 1 1 Managing 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 3 in which we identify a set of buttons a plot of the activity levels of the selected reactants and a time slider The buttons above the graph have the following purposes Change title allows to select a new title for the tab this can be useful e g when comparing different simulations
15. named A_StdDev for ANIMO to recognize it and properly display the data series with the associated error values 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 10 parameter settings of Section 2 1 in 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 2 and that the inactivating interaction in the loop is faster than the ones activating the target node Once a satisfying profile is obtained the parameters can be rescaled in groups or all at the same time This allows us to easily match a model against experimental data that show a similar profile on a different time scale e g the model produces a peak similar to the one in the data but 20 minutes too early In ord
16. nload 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 amp 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 19 or home myuser programs uppaal 4 1 19 ae 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 19 the two commands to be entered are d 22 cd user Programs uppaal 4 1 19 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 clo
17. odes 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 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 1 nodes that will be plotted have a blue border 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 All edges connecting disabled nodes are automatically disabled Each plot in an ANIMO Results 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 a centre click on 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 graph 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
18. 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 If the model is non deterministic i e its evolution is not expected to be exactly the same for every single simulation run see the uncertainty settings in Options Section 2 6 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 shading 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 replicated experimental data In a csv file the column containing the standard deviation values for column A should be
19. ress Update Selected or just press Update All to update all apps Cytoscape will download and install the new versions of the apps 2 6 ANIMO Options In the Control Panel the ANIMO tab contains also an Options button it is located below the Legend Pressing that button will open the options dialogue for ANIMO where the following configuration settings can be changed location of the verifyta executable the location of verifyta can be changed if for example a wrong location was previously selected UPPAAL was not already installed when ANIMO was run for the first time or a new version of UPPAAL has been installed uncertainty in reaction parameters a degree of uncertainty can be set for the k parameters of all the reactions in ANIMO models introducing some non determinism in the simulations For example if a value of 5 uncertainty is chosen a given reaction that would normally take exactly 10 seconds to perform will occur after a number of seconds randomly chosen from a uniform distribution in the closed interval 9 95 10 05 The default value for uncertainty is 5 so reaction times are as described by default In order to get a deterministic model especially useful to make model checking much easier for the computer set this parameter to 0 i e uncheck the option type of underlying Timed Automata model two types of Timed Automata models are avail able reaction centred and reactant centred The latter is in general more effic
20. scription of such tools in Subsection 2 2 or when the data series was loaded from a csv file which contains error estimates see instructions on how to prepare csv files including error estimation data series in Subsection 2 2 The Change plotted series button allows to select which nodes of the network are displayed in the graph initially only nodes marked as plotted when a simulation is performed are included in the graph with this button it is still possible to change the set of plotted nodes without the need to perform a new simulation 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 represented with different node colouring in the Network window on the left For an example see Figure 3 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 Finally the Copy button beside the slider _ can be used to set the activity levels in the network to the time point selected with the slider This is useful if for example we want to change some conditions from a certain point in a simulation 2 1 2 Editing a network in Cytoscape Nodes and arcs can be placed in the network as shown previously right click on an empty space in the Network window
21. se that window Installing ANIMO 1 ANIMO is free only for academic use For commercial licenses please contact us at s schivo ewi utwente nl Run Cytoscape Click the menu command Apps App Manager the App Manager window will open Click on ANIMO in the list of available apps and then on the Install button ANIMO will be automatically downloaded and installed 5 You can close the App Manager window 6 ANIMO will automatically try to locate the verifyta executable included in UPPAAL which is needed to verify Timed Automata models If you know the location where you installed UPPAAL you can just stop the process press on the small X on the right and indicate the location of the verifyta executable You can find it where it was copied at step 5 in Section 1 3 ay A in the bin bin Linuax bin Win32 depending on your operating system directory inside the UPPAAL installation directory where it was unzipped at step 4 in Section 1 3 7 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 L 2 8 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 Session Answer pos
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