Introductions

What about you?

• Clinicians
• Bench Biologists
• Bioinformaticians
• Computer Scientists
• Chemists
• Mathematicians
• Other

Introductions

Prior Cytoscape or network analysis experience?

• None -- that's what I'm here for!
• Some network analysis, but what's Cytoscape?
• I've played with Cytoscape, but still have a lot to learn.
• I use Cytoscape all the time...
  (hey, I could probably teach this course!)

Networks as Tools

Networks as Tools

Applications in Research

Applications in Research

• In 2010, a Nature Methods paper introduced major themes of network visualization.
• (a) Mass spec analysis: 400 PPI in pneumonia microbe.
• (b) Subnetwork with function annotations.
• (c) Layout with knowledge of complexes.
• (d) Collapsed metanodes to reduce complexity.

Applications in Research

• Study of interactions with ASD-associated proteins.
• Y2H with human protein fragments of 26 ASD proteins: 848 interacitons with 539 proteins.
• Merged with known interactions from public datasets.

Applications in Research

• SARS-CoV-2-human interactions.
• AP-MS with 26 SARS-CoV-2 proteins: 332 interactions human proteins.
• Merged human-human PPI datasets to identify complexes.
• Used fill color to highlight known drug targets.

Applications in Research

• Genetic interactions for the entire yeast genome.
• Synthetic lethal screen of 1712 genes: 170,000 interactions.
• Visualization of clustering within and between functional groups annotated by GO.
• Predict functions and identify regulatory subnetworks.

Applications in Research

• Molecular profiling of two types of brain tumors.
• GSEA to identify distinguishing gene sets (nodes).
• Functional annotation of a network of gene sets.

Applications in Research

• TCGA's seriers of molecular cancer profiles.
• Clinical, genomic, methylation, RNA and proteomic signatures.
• Multiple data types integrated onto signaling network.
• Includes patient sample-level data.

Applications in Research

Core Concepts

Networks and Tables

Networks
e.g., PPIs or pathways

Tables
e.g., data or annotations

Visual Styles

Core Concepts

Cytoscape Apps!

apps.cytoscape.org

Loading Network Data

• Cytoscape can import network data from:
  • Files (or URLs)
    • Excel, TSV, CSV
    • XGMML: eXtensible Graph Markup and Modelling Language
    • SBML: Systems Biology Markup Language
    • BioPAX
    • PSI-MI
    • SIF: Simple Interaction Format
    • GML: Graph Markup Language
    • ... and others depending on loaded Apps

Loading Network Data

• Cytoscape can import network data from:
  • Public repositories:
    • PSICQUIC
    • STRING (via the stringApp)
    • IntAct (via the IntActApp)
    • Reactome (via the ReactomeFI app)
    • WikiPathways (via the WikiPathways app)
    • Pathway Commons (via the CyPath2 app)
    • NDEx
  • Automation:
    • Command line scripts
    • CyREST via R, Python, etc

Loading Table Data

• Cytoscape can load tables from:
  • Files (or URLs)
    • Excel, TSV, CSV
  • Public repositories
    • BioMart
  • Automation:
    • Command line scripts
    • CyREST via R, Python, etc

Saving and Exporting

• Sessions save everything as .cys files:
  Networks, Tables, Styles, Screen sizes, etc.
• Export networks in different formats:
  SIF, GML, XGMML, BioPAX, JSON
• Export tables as CSV files
• Publication quality graphics in several formats:
  PDF, SVG, PNG, and JPEG

Tour of Cytoscape

2. Open CyBrowser
3. Enter URL: tutorials.cytoscape.org

Tour of Cytoscape

4. Click on Tour of Cytoscape
5. Resize window to your preference

Biological Network Taxonomy

Interactions
• Protein-Protein
• Protein-Ligand
• Domain-Domain
• Others
  • Residue or atomic
  • Cell-cell
  • Epidemiology
  • Social networks

Biological Network Taxonomy

Similarity
• Protein/Sequence Similarity Networks (PSNs or SSNs)
• Chemical similarity
• Ligand similarity (SEA)
• Others
  • Tag clouds
  • Topic maps

Biological Network Taxonomy

Pathways
• Metabolic
• Signaling
• Regulatory
• Disease

Networks
• Interaction
  • Physical, Genetic, Social
• Similarity
  • Structural, Sequence, Topical

Experimental Techniques

High throughput
• Yeast 2 hybrid

Experimental Techniques

High throughput
• Yeast 2 hybrid
  • Pros:
    • Relatively low-tech
    • Scalable
  • Cons:
    • High false positive and false negative rate

Experimental Techniques

High throughput
• Yeast 2 hybrid
• Tandem Affinity Purification/Mass Spectrometry (AP/MS)

Experimental Techniques

High throughput
• Yeast 2 hybrid
• Tandem Affinity Purification/Mass Spectrometry (AP/MS)
  • Pros:
    • Quantitative results in vivo
  • Cons:
    • Tag can obscure binding
    • Protease can cleave protein
    • Not good for transient interactions

Experimental Techniques

High throughput
• Yeast 2 hybrid
• Tandem Affinity Purification/Mass Spectrometry (AP/MS)
• BioID - Proximity-dependent Biotinylation

Experimental Techniques

High throughput
• Yeast 2 hybrid
• Tandem Affinity Purification/Mass Spectrometry (AP/MS)
• BioID - Proximity-dependent Biotinylation
  • Pros:
    • Can be used for membrane-bound complexes and insoluble proteins
    • Can be used to capture a "snapshot" of dynamic processes
  • Cons:
    • Can capture random collisions (false positives)
    • Size of BirA* (321 AA can interfere with binding (false negatives))
      • NOTE: BioID2 is smaller (233 AA)
    • 10 nm labeling radius. Need to consider location of fusion

Experimental Techniques

High throughput
• Yeast 2 hybrid
• Tandem Affinity Purification/Mass Spectrometry (AP/MS)
• BioID - Proximity-dependent Biotinylation
  • BioID2 - same procedure as BioID, but uses a smaller promiscuous biotin ligase
  • split-BioID - like BioID, but BirA* is split into CBirA* and NBirA* parts, which are fused in the presence of Rapamycin, providing a conditional method
  • APEX - a similar technique that uses an engineered ascorbate peroxidase, but is most often used to label as many cells as possible in an organelle rather than protein-protein interactions.

Experimental Techniques

High throughput
• Yeast 2 hybrid
• Tandem Affinity Purification/Mass Spectrometry (AP/MS)
• BioID - Proximity-dependent Biotinylation
• Protein-fragment complementation assays
• Phage display
• Protein microarrays
• Chemical crosslinking

Jensen & Bork, Science, 2008

Experimental Techniques

High throughput
• Genetic interactions
  • Measures functional linkage between genes
  • Orthogonal to physical interaction techniques
  • Two approaches
    • Synthetic Genetic Array (SGA)
    • Epistatic MiniArray Profile (E-MAP)

Experimental Techniques

Experimental Techniques

Experimental Techniques

High throughput
• Genetic Interactions: Epistatic MiniArray Profile (E-MAP)
  • High throughput
  • Quantitative
  • Can provide corroboration to PPI data

Experimental Techniques

Low throughput
• Xray crystallography
• NMR
• SAXS
• ELISA Binding Assays
• Co-IP

Computational Techniques

• Text mining
  • Search literature for protein-protein co-occurrences
• Orthology
  • Predicts interaction based orthologous pairs in another species
• Domain pairs
  • Predicts interaction based on domains interacting in other proteins

Public Repositories

• Pathway data
  • WikiPathways
  • Reactome
  • KEGG
  • Pathway Commons (collection)
  • Published Pathway Figures

Public Repositories

• Network data
  • STRING
  • IntAct
  • NDEx (collection)

Sources of PPI Data

• Experimental Techniques
  • High Throughput
  • Low Throughput
• Computational Techniques
• Public Repositories

Know what to look for.
Know what you're getting.

Starting with a gene list......

Networks

• STRING (stringApp)
• NDEx (built-in)
• PSICQUIC (built-in)
• IntAct (IntAct App)

STRING

stringApp

STRING Settings

STRING Results

STRING Options

STRING Enrichment

NDEx

• Repository for biological network knowledge
• Organisations and individual scientists can deposit
• Use CyNDEx App to import/export networks
• Featured collections include:
  • Pathway Interaction Database (NCI-PID)
  • Cancer Cell Maps Initiative (CCMI)
  • The NDEx Butler
  • NetPath

NDEx Search Results

NDEx Network

PSICQUIC

PSICQUIC search results

• Select results from multiple sources
• Interaction type is included

IntAct

IntAct App

IntAct

Summary View

IntAct

Evidence View

IntAct

Mutation View

IntAct

Filters

• Can filter network by:
  • MI Score
  • Interaction detection method
  • Participant detection method
  • Host organism
  • Expansion (e.g. spoke vs. matrix)
  • Type (interaction type)
  • Mutations (hides unmutated edges)

Pathways

• WikiPathways (WikiPathways App)
• Reactome (ReactomeFI App)
• Pathway Commons (CyPath2 App)
• KEGG (KEGGscape App)

WikiPathways

• Handmade pathway models
• Collaborative platform open to all researchers
• Thousands of pathways for dozens of species

WikiPathways Search

WikiPathways Results

WikiPathways Results

Pathway Database Apps

Depiction

• Various ways to depict biological networks:
  • Node-Link (graph) representation
  • Partitioned Node-Link representation
  • Matrix representation
    • Can be useful for very dense networks
    • Can also map information into cells of matrix
      • e.g. degree, color scale (heat map)

Data Mapping

• Mapping of data values associated with graph elements onto graph visuals
• Visual attributes
  • Node fill color, border color, border width, size, shape, opacity, label
  • Edge type, color, width, ending type, ending size, ending color
• Mapping types
  • Continuous (numeric values)
  • Discrete (categories)
  • Passthrough (labels)

Data Mapping

• Avoid cluttering your visualization with too much data
  • Highlight meaningful differences
  • Avoid confusing the viewer
  • Consider creating multiple network images

Layouts

• Layouts determine the location of nodes and (sometimes) the paths of edges
  • Types:
    • Simple
      • Grid
    • Hierarchical
      • layout data as a tree or hierarchy
      • Works best when there are no loops
    • Circular (Radial)
      • arrange nodes around a circle
      • could use node attributes to govern position
        • e.g. degree sorted

Layouts

• Types:
  • Force-Directed
    • simulate edges as springs
    • may be weighted or unweighted
  • Combining layouts
    • Use a general layout (force directed) for the entire graph, but use hierarchical or radial to focus on a particular portion
  • Multi-layer layouts
    • Partition graph, layout each partition then layout partitions
  • Many, many others

Layouts

• Manual tweaks with Node Layout Tools
• Scale
• Align
• Rotate

Layouts

• Use layouts to convey the relationships between the nodes.
• There is not one correct layout. Try different things.
• Layout algorithms may need to be “tuned” to fit your network.

Animation

• Animation is useful to show changes in a network:
  • Over a time series
  • Over different conditions
  • Between species

Advanced Visualization in Cytoscape

Typically, we don't have enough time to discuss all of the capabilities of Cytoscape visualizations. This module tries to fill in some of the gaps. Here are the topics we're going to cover:

• More about styles
• Working with text
• Controlling node position
• Adjusting the view (Node Position Tools)
• Tuning layouts
• Graphical annotations (marks)
• Working with palettes
• Add graphics to nodes
• Animations made easy
• Using Charts to visualize enrichment results

Advanced Visualization in Cytoscape

1. More about styles

• There are many styles that are not shown by default
• Style columns are Def. (default), Map. (mapping), and Byp. (bypass)
• Bypass is used to override (bypass) both the mapping (if any) and the default
• To set a node or edge bypass
  1. select the nodes or edges you want to set the visual property
  2. click on the bypass column for the visual property you want to set
  3. set the value as appropriate
• Now, those properties will be permanently set for those nodes or edges
• To clear the value, select the nodes or edgges again, right-click on the bypass column and select Remove Bypass

Advanced Visualization in Cytoscape

1. More about styles

• Column styles:
  • Column styles control the way data values are shown in the various tables (node, edge, network)
  • Like node and edge styles, column styles can be mapped using column data values
  • In order to use column styles, you need to add each column and then set the style for that column

Try it!

Advanced Visualization in Cytoscape

2. Working with fonts

• Cytoscape has several label/font properties that are not shown by default
• The following properties are available:
  • Label: usually used to set up a passthrough mapping for the label text
  • Label Autorotate: (for edges only) rotate the label to be parallel with the edge
  • Label Font Face: this is the font face to use -- usually a very long list
  • Label Font Size: the font size, can also be a passthrough mapping
  • Label Position: where the font should be placed
  • Label Transparency: the transparency of the font
  • Label Width: the maximum width of the label. Labels longer than this will be wrapped
  • Label Rotation: the angle of the text in degrees
  • Label Background Color: the color of the text background
  • Label Background Shape: the shape of the text background
  • Label Background Transparency: the transparency of the text background
• Label position and rotation can also be done interactively for node and edge labels.

Advanced Visualization in Cytoscape

2. Working with fonts

Try it!

Advanced Visualization in Cytoscape

3. Controlling node and edge position (depth)

• Nodes and edges with the same Z order are drawn at the same time
• This can lead to important information being obscured
• Z order can be set by the Z Location visual property
• Nodes or edges with larger Z are drawn closer to the viewer (on top)
• Setting lots of different Z Location values can slow down graphics performance
• Best approach is to use a few different Z Location values or to set it manually using a Bypass

Advanced Visualization in Cytoscape

3. Controlling node and edge position (depth)

Try it!

Advanced Visualization in Cytoscape

4. Adjusting the view

• Node layout tools (Layout→Node Layout Tools) provide important tools to help "tweak" the final look of a network

Advanced Visualization in Cytoscape

5. Tuning layouts

Advanced Visualization in Cytoscape

5. Tuning layouts

Try it!

Import galFiltered.sif

Play with both the node layout tools and layout tuning

Advanced Visualization in Cytoscape

6. Working with annotations (marks)

• Cytoscape has three separate drawing surfaces:
  • Network canvas (where nodes and edges are drawn)
  • Background canvas (drawing surface behind the nodes and edges
  • Foreground canvas (drawing surface in front of the nodes and edges
• Annotations are drawn on either the foreground or background canvases
• Annotations are exported as objects, so are high quality graphically^*
• Annotation types:
  • Shapes, Text, Bounded Text, Images, Arrows
  • Groups, which group annotations together
• The Annotation Panel makes working with annotations easy and intuitive!

Advanced Visualization in Cytoscape

6. Working with annotations (marks)

Try it!

Advanced Visualization in Cytoscape

7. Palettes

• Cytoscape now supports palettes for choosing colors
• Three palette types are supported:
  • Sequential: used for gradients with only positive or negative values
  • Divergent: used for gradients with both positive and negative values
  • Qualitative: used for discrete color mapping
• The following palettes are provided by default:
  • ColorBrewer: provides all three palette types
  • Viridis: provides sequential palettes
  • Built-in: built-in Rainbow, Rainbow OSC, and Random qualitative palettes, and a variety of built-in divergent palettes
• Palettes are also used to select colors, when a current palette is defined

Advanced Visualization in Cytoscape

8. Adding graphics to nodes

• Sometimes, we want to map graphics onto nodes
• This should be done very sparingly
• Using Cytoscape's built-in charts
  • Image/Chart 1 is included by default
  • Additional charts can be added via Properties→Paint→Custom Paint...
  • Mappings aren't really supported for charts, so select either the Default, or (if one or more nodes are selected, by Bypass) column.
  • This will bring up the Graphics panel
  • Click on Charts or Gradients as desired

Advanced Visualization in Cytoscape

8. Adding graphics to nodes

• enhancedGraphics was written before Cytoscape charts were added to the core
• The format is not nearly as convenient, but allows for greater flexibility and specificity
• Adding a chart to a node is simple:
  1. Create a new text column in the node table
  2. Add the chart instructions (see below for samples)
  3. Set up a visual style that uses the column you created as a passthrough mapping for one of the CustomGraphics styles.

Advanced Visualization in Cytoscape

8. Adding graphics to nodes

• Some example chart instructions:
  • Basic charts:
    • linechart: colorlist="up:blue,down:red" attributelist="Values"
    • piechart: attributelist="Values" colorlist="contrasting" labellist="First,Second,Third,Fourth,Fifth"
    • piechart: attributelist="a,b,c,d" colorlist="red,green,blue,yellow"
    • barchart: attributelist="a,b,c,d" colorlist="up:yellow,down:green"
    • stripechart: colorlist="red,white,blue"
    • heatstripchart: separation="1" valuelist=".2,-1,3,1" labellist="cond1,cond2,cond3,cond4"
  • Circos charts:
    • circoschart: firstarc=.5 arcwidth=.4 colorlist="red, green, blue, white" showlabels=false attributelist="Circle1,Circle2"
    • circoschart: arcstart=90 firstarc=.5 arcwidth=.3 range="-5,5" colorlist="up:yellow,down:blue,zero:black" valuelist="1,1,1,1" showlabels=false attributelist="Circle1,Circle2"
  • "Label" charts:
    • label: attribute=name labelsize=10 outline=true background=true color=white dropShadow=true

Advanced Visualization in Cytoscape

8. Adding graphics to nodes

• Apps that create custom paints:
  • chemViz2: draws chemical structure onto nodes
  • enhancedGraphics: used to draw charts and labels onto nodes
  • stringApp: uses custom paint for the "glass sphere" look, calls chemViz2 for chemical drawings, enhancedGraphics for "fancy" labels
  • OmicsVisualizer: uses enhancedGraphics for rings of data

Advanced Visualization in Cytoscape

9. Animations made easy

• The CyAnimator app is a key frame animation tools for Cytoscape
• To use, bring up the interface (Apps→CyAnimator)
• Add a frame to the animation by clicking the + button

Advanced Visualization in Cytoscape

10. Using Charts to visualize enrichment results

• The Enrichment Table App provides the functionality of functional enrichment analysis for any network loaded into Cytoscape.
• To use, bring up the interface (Tools→Enrichment Table→Perform Gene Enrichment)
• Add a chart to the network by clicking the Draw Chart button

Advanced Visualization in Cytoscape

Try it out!

Advanced Visualization in Cytoscape

Questions?

Additional examples: Step-by-step protocols

Working with Groups in Cytoscape

Cytoscape groups are:

• Collections of nodes (no additional enforced semantics)
• May be hierarchical (i.e. a group can be a member of another group)
• May share nodes, however:
  • Compound node visualization won't work properly
  • Expand/collapse are not determnistic, so shared nodes might be included or not
• Groups are nodes themselves!

Working with Groups in Cytoscape

Manipulating groups

• Create a group:
  • Highlight a group of nodes
  • Right-click over a node or empty space
  • Select Group→Group selected nodes
• Collapse a group:
  • Double-click on any member of the group
• Expand a group:
  • Double-click on the collapsed group node
• Delete a group:
  • Right-click on the group, or any node in the group
  • Select Group→Ungroup Selected Nodes

Working with Groups in Cytoscape

Some apps that create groups

• setsApp
  • Define sets of nodes by selection, attribute value, or by importing them
  • Right-click on the set and select Create group from set nodes
  • Set aware layouts are also available
• clusterMaker2
  • Provides the option to create groups from network clusters

Working with Groups in Cytoscape

Group visualization

• None: no visualization for the group. Grouped nodes look the same as other nodes.
• Compound Node: visualize the group as a compound node, essentially a node (the group) that contains other nodes (the members of the group). Individual member nodes can be individually selected and manipulated.
• Show Group Node: the group node itself is added to the network and "membership edges" are added between the group node and each group member
• Single Node: similar to compound node in that all of the member nodes are visible, but member nodes can not be individually selected or manipulated. Essentially, the entire group is treated as a single node.

Working with Groups in Cytoscape

• Cytoscape groups can optionally aggregate member node data.
• Aggregations can occur on all columns, or only specified columns
• Aggregation options:
  • Numeric columns: None, Average, Minimum value, Maximum value, and Sum
  • String columns: None, Comma-separated Values, Tab-separated Values, Most Common Value, and Unique Values
  • Boolean columns: None, Logical AND, and Logical OR
  • List columns:
    • For numeric lists, same options as numeric columns plus Concatenate and Unique Values
    • For String lists, None, Unique Values and Concatenate

Working with Groups in Cytoscape

Configuring Groups

Groups are configured using the Group Preferences dialog (Edit→Preferences→Group Preferences)

Working with Groups in Cytoscape

Try it out!

Open up Cytoscape, and select the Ivacaftor Coauthor network. Play around with grouping either by creating groups manually or by installing the setsApp and creating groups based on the node type column.

"Exam"

To make sure you now have a working knowledge of Cytoscape, choose one of the following datasets to analyze in Cytoscape:

• Colins (2007): A merged dataset of three yeast APMS experiments. Find the putative complexes.

Thank You!