healthmedicinet

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.

Figure 1. NetworkPainter enables researchers to quickly draw publication quality signaling diagrams.
PBMC immune signaling diagram created with NetworkPainter. Diagram adapted from Bodenmiller
et al., 2012 Figure S6 10]. Observed and unobserved signaling nodes are colored dark and light red, respectively;
extracellular receptors are colored green.

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.

Figure 2. NetworkPainter visualizes multi-parameter data in the context of biological pathways.
PBMC pathway painted with a time course of mass cytometry measurements obtained at
0, 1, 5, 15, 30, 60, 120, and 240 min post-LPS induction 10]. Animated pathway diagram highlights the differential cell type responses observed
by Bodenmiller et al. Heatmaps indicate each node’s median activity across the fourteen
observed cell types (first row: CD14- monocytes; second row: CD14+ monocytes; third
row: dendritic cells, IgM+ B cells, IgM- B cells, NK cells; last row: CD8+ T cells,
CD4+ T cells). Nodes are colored by their mean value across all fourteen cell types.
Yellow color indicates high activity; blue indicates low activity. An interactive,
animated version of Figure 2 is available at http://covert.stanford.edu/networkpainter/KarrEtAl2014Fig2.

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.