NetworkPainter provides researchers a web-based graphical interface to visually analyze cytometry data. NetworkPainter only requires a web browser with Adobe Flash Player. Below we briefly describe how to use NetworkPainter. Additional file 1 provides further information about how to use the software.
First, researchers use the graphical interface to draw a pathway diagram, formalizing their prior biological knowledge about their pathway, including its molecular components and their interactions. Users simply drag and drop molecules to add them to the diagram and assign them to subcellular compartments. Users can draw arrows by selecting two molecules and selecting “Draw arrow” from the right-click context menu. Users have full control over the graphical appearance of each molecule and compartment including its color, shape, and size. Twenty-six shapes are available including polygons, as well as several commonly used graphical representations of DNA, RNA, and protein. Figure 1 depicts a PBMC immune signaling diagram created with NetworkPainter.
To help users create visually pleasing diagrams, NetworkPainter provides an automatic layout tool to improve the arrangement and spacing of molecules in diagrams. In addition, users can import diagrams from the KEGG PATHWAY database [33] or from diagrams published by other users. Furthermore, NetworkPainter provides basic editing features including cut, copy, paste, undo, redo, auto save, find, zoom, and pan.
Second, users upload and annotate experimental data. Cytobank users use Cytobank’s existing graphical interface to create an experiment, upload flow or mass cytometry data, and annotate the individual, cell type, condition, dosage, channel, and timepoint of each measurement. Standalone users can upload data using the JSON format described in the online help.
Third, users link diagram nodes with experimental measurements and perturbations. Researchers use drop-down lists to select the experimental channel corresponding to each observed signaling molecule. Optionally, researchers can also use drop-down lists to enter the enhansive or repressive effect of each experimental perturbation.
Fourth, researchers can specify how to display the experimental individual, cell type, condition, and dosage dimensions of their data. Researchers can select two dimensions to display in small heatmaps below each experimentally linked signaling molecule. These heatmaps display the measurement of the corresponding experimental channel for each value of the two selected heatmap dimensions at each timepoint, averaged over the unselected heatmap dimensions. Optionally, NetworkPainter can cluster and order the selected heatmap dimensions using hierarchical agglomerative clustering [34] and optimal leaf ordering [35]. A legend and tooltips indicate the data plotted in each heatmap cell. These heatmaps are designed to help researchers visualize more of their data simultaneously, as well as compare measurements across individuals, cell types, conditions, and dosages.
Fifth, researchers can specify the averaging algorithm NetworkPainter uses to collapse the experimental data displayed in the signal molecules and heatmaps. Users can select among all of the statistics (e.g. mean, median, min, max), equations (e.g. raw, log, fold, arcsinh, difference), and controls (e.g. first row, first column, table min, table max) available in Cytobank’s illustrations. Researchers can also select the color mapping between the experimentally observed values and painted colors. The online help lists all of the available statistics, equations, controls, and colormaps.
Sixth, researchers can use the playback controls at the bottom-left of the diagram to dynamically paint each experimentally perturbed or observed signaling molecule over the measured timecourse. After clicking the play button, NetworkPainter colors each experimentally linked signaling molecule according to the measured value of the corresponding experimental channel at the current timepoint and the selected colormap, averaged over all other experimental dimensions. NetworkPainter interpolates timepoints to produce smooth animations. Unobserved nodes are colored grey and optionally, the compartments and membranes are colored grey to emphasize the dynamics of the observed nodes. Optionally, small heatmaps below the signaling molecules enumerate the values of the experimental channel across the two selected heatmap dimensions. Users can also control the playback speed and looping. Figure 2 depicts eight static snapshots of a human PBMC immune signaling diagram painted by NetworkPainter with the first reported mass cytometry timecourse containing fourteen measured signaling nodes [10]. An interactive, animated version of Figure 2 is available at http://covert.stanford.edu/networkpainter/KarrEtAl2014Fig2. The case study below describes how NetworkPainter can be used to analyze this data.
Seventh, researchers can save diagrams to either the Cytobank or standalone server for later use and/or sharing with collaborators. Cytobank users can give collaborators permission to view or edit diagrams by granting permission to their associated experiments. Standalone users can use a simple graphical interface to grant read, write, or administer privileges to other users. Once collaborators are granted permission, they can edit a diagram. However, each diagram can only be edited by a single user at a time. Both Cytobank and standalone users can also publish diagrams to share them with all users.
Lastly, researchers can export diagrams to several image (GIF, JPEG, PNG, SVG) and animation (GIF, SWF) formats for use in papers, presentations, and websites. NetworkPainter also exports diagrams to the Biological Pathway Exchange (BioPax), CellML, and Systems Biology Markup Language (SBML) standards for use with other network analysis software programs, as well as to a JSON format which can be subsequently imported back into NetworkPainter. In addition, NetworkPainter can generate MATLAB scripts for Boolean simulations.