Integrative Bioinformatics using Cytoscape (and R2)

(Bio)Chemistry Concentrations Molecular structures Reaction equations Quantitative Defined experimental setup

(Bio)Chemistry versus Molecular Biology some basic concepts

Molecular Biology Regulation Large biomolecules Large scale processes Qualitative Complex experimental setup (by necessity!)

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Molecular Biology: New techniques Integrative Bioinformatics needed

(Deep)Sequencing Arrays Proteomics Quantitative analysis
handling large datasets statistics

Capturing complexity
integration graphs

Integrative Bioinformatics: Integrated Bioinformaticians!

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Integrative Bioinformatics: An example

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Integrative Bioinformatics: What they did

1. Sequence genome; assign gene function using protein sequence, structural similarities (Bonneau et al., 2004; Ng et al., 2000) 2. Perturb cells: environmental factors; knockouts (Baliga et al., 2004; Kaur et al., 2006; Kottemann et al., 2005) 3. Measure changes: microarrays (Baliga et al., 2004;Kaur et al., 2006; Whitehead et al., 2006). 4. Integrate diverse data (mRNA levels, evolutionarily conserved associations among proteins, metabolic pathways, cis-regulatory motifs, etc.) with the cMonkey algorithm to reduce data complexity and identify subsets of genes that are coregulated in certain environments (biclusters) (Reiss et al., 2006). 5. Using the machine learning algorithm Inferelator construct a dynamic network model for influence of changes in EFs and TFs on the expression of coregulated genes (Bonneau et al., 2006). 6. Explore the network with Gaggle, a framework for data integration and software interoperability to formulate and then experimentally test hypotheses to drive additional iterations of steps 26 (Shannon et al., 2006)

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Integrative Bioinformatics: Their framework

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Integrative Bioinformatics: results

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Goes to show that:

1. Aggregate 2. Search/Visualize 3. Analyze/Feedback Combine data from different sources Filter Algorithms

Need for adaptable software

Goal: Facilitate ideas

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Cytoscape - Network Visualization and Analysis

Freely-available (open-source, java) software, easily extensible (Plugin API) Visualizing networks (e.g. molecular interaction networks) Analyzing networks with gene expression profiles and other cell state data (GO, proteomics, ) Used in several hundred analyses in recent literature Continuity guaranteed

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An example Cytoscape work-flow

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Cytoscape Workflow
1. Load Networks (Import network data into Cytoscape) 2. Load Attributes (Get data about networks into Cytoscape) 3. Analyze and Visualize Networks 4. Prepare for Publication A specific example of this workflow:
Cline, et al. Integration of biological networks and gene expression data using Cytoscape, Nature Protocols, 2, 2366-2382 (2007).

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Networks as graphs
A Network is a collection of
Nodes (or vertices) Edges connecting nodes (directed or undirected, weighted, multiple edges, self-edges) Nodes can represent proteins, genes, metabolites, or groups of these (e.g. complexes) - any sort of object Edges can be either physical or functional interactions, activators, regulators, reactions - any sort of relations

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Cytoscape Workflow
1. Load Networks (Get network data into Cytoscape) 2. Load Attributes (Get data about networks into Cytoscape) 3. Analyze and Visualize Networks 4. Prepare for Publication

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Creating a network

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Free-format Text and Excel Files

Specify Input File
Define Columns

Text Parsing Options

Preview

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Pathways: plenty resources

http://pathguide.org : over 240 pathwaydbs

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All kinds of network data

Physical interactions
Protein Protein interactions Protein DNA interactions Metabolic interactions

Functional interactions
Co-expression relations Genetic interactions Knockout/siRNA targets
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Pre-formatted Network Files

Cytoscape supports many popular file formats:
SIF (Simple Interaction Format) GML (Graph Markup Language) XGMML (eXtensible Graph Markup and Modeling Language) BioPax (Biological Pathway Data) PSI-MI 1 & 2.5 (Protein Standards Initiative) SBML Level 2 (Systems Biology Markup Language)

Available for download from data sources (URLs, web-services, formatted table files)
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Internet Databases
Cytoscape version 2.6
web service clients: import networks directly from several trusted internet resources IntAct (MBL-EBI) PathwayCommons (collection of data resources) NCBI Entrez Gene Many more will be included...

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Interaction Database Search

Import Visualize and Analyze

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Cytoscape Workflow
1. Load Networks (Get network data into Cytoscape) 2. Load Attributes (Get data about networks into Cytoscape) 3. Analyze and Visualize Networks 4. Prepare for Publication

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What are Attributes?

Any data that describes or provides details about the nodes and edges in the network
Gene Expression Data Mass Spectrometry Data Protein Structure Information Gene Ontology (GO) terms Interaction Confidence Values, etc

Cytoscape support multiple data types

Numbers (integers, floats) Text (strings) Human Genetics Logical (booleans)

Attribute Management
Select Attributes for Display

Node or Edge ID

Strings and floating type of attributes

Specific Attribute Tabs

Load Attributes: Import Attribute Files

Map data about Networks onto Networks. Attributes can be loaded in many of the same ways as networks.
Import pre-formatted attribute files Import formatted text or Excel files Create attributes manually in attribute editor Load attributes from web services ID mapping though node attributes
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ID Mapping
Mapping identifiers from one source to another is a major challenge Multiple levels of IDs E.g. probe>gene ->peptide->protein Cytoscape provides an ID mapping through the BioMart web service of EBI to convert the IDs Not perfect but sufficient Additional mapping mechanism underway

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Cytoscape Workflow
1. Load Networks (Get network data into Cytoscape) 2. Load Attributes (Get data about networks into Cytoscape) 3. Analyze and Visualize Networks 4. Prepare for Publication

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Visual Data Integration

1. Network Data
YDR382W YDR382W YFL039C YFL039C pp pp pp pp YDL130W YFL039C YCL040W YHR179W

VizMapper

2. Attribute Data
ExpressionValue YCL040W = 0.542 YDL130W = -0.123 YDR382W = -0.058 YFL039C = 0.192 YHR179W = 0.078

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VizMapper
List of Visual Styles Default Visual Style Editor List of Visual Attributes

List of Data Attributes

Mapping definition

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Types of mappings
Continuous Continuous Data mapped to Continuous Visual Attributes (e.g. gene expression levels mapped to node color) Continuous Data mapped to Discrete Visual Attributes (e.g. p-value categories mapped to node shape) Discrete Discrete (categorical) Data to Discrete Visual Attributes (e.g. GO annotation mapped to node shape) Discrete Data mapped to Continuous Visual Attributes(e.g. multiple GO terms mapped to pie coloring)

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Network Filtering

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Several Layout Algorithms

Spring-embedded
Circular Hierarchical

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Linkout
Nodes and Edges act as hyperlinks to external databases. Userconfigurable URLs Collection of the biological results

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Cytoscape Workflow
1. Load Networks (Get network data into Cytoscape) 2. Load Attributes (Get data about networks into Cytoscape) 3. Analyze and Visualize Networks 4. Prepare for Publication

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Prepare for Publication

Fine tune the Figures Manual Layout manipulation options (align, scale, rotate) Manually override visual styles

place labels, change colors, etc.

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Finalizing the Figures

Publication Quality Graphics in several formats
PDF, EPS, SVG, PNG, JPEG, and BMP

Export Session to HTML for Web

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Cytoscape: So what?
The big Pro Cyto argument: EXTENSIBLE Plugins, Plugins, Plugins
In our case enabled extended array data analysis

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Cytoscape is Extensible
Cytoscape is open source and free software A plugin interface that allows any programmer to write their own extensions to Cytoscape Plugins represent the primary biological analysis mechanism in Cytoscape Plugins are distributed from a central Cytoscape database and can be installed while running
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Hello World Plugin

http://cytoscape.org/cgi-bin/moin.cgi/Hello_World_Plugin http://cytoscape.org/cgi-bin/moin.cgi/Developer_Homepage
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Extending the workflow through plugins

Graph based integration and analysis of molecular biological data

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Integrative Bioinformatics in our group

Aggregate data: 18000+ Affymetrix arrays Tumor series Public data Experiments Manipulate celllines; Lentiviral library Search/Visualize/Selection: R2 Statistical cutoffs Correlations: R2 Clinical data coupling Analysis/Feedback: R2 and Cytoscape Known Interactions Transcription Factor binding

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Integrative Bioinformatics in our group

Patient data GEO arrays HGServer R2-array analysis interface
Statistical analysis Perl module

DB

Cytoscape webstart

AMC Plugin

Cytoscape interface

External data sources Array data: Tumor and Experiments

Canonical paths

Human Algorithms Genetics

Array data analysis: R2

Mainly work by Jan Koster

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R2 interface: Demo

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R2 interface

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R2 interface

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R2 interface

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R2 interface

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R2 interface

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Timeseries in R2 / Cytoscape (Demo)

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Timeseries in R2

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Timeseries in R2

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Timeseries in R2 Integration with Cytoscape through webstart

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Timeseries in Cytoscape: Visualization

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Timeseries in Cytoscape: Aggregate data

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Timeseries in Cytoscape: Search/Filter

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Timeseries in Cytoscape: Filter

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Timeseries in Cytoscape

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Timeseries in Cytoscape

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Tf (green) and partners (red)

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Filtering

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Filtering

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Coloring, layout

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Resuming:
1. Aggregate 2. Search/Visualize 3. Analyze/ Feedback Combine NOTCH3 knockout data with TF and PPi data Layout timeseries/Find downstream targets Identify MSX1/Knockout in new experiment

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More Plugin Examples

BiNGO (Enriched GO categories found in the sub-network) WikiPathways (Visualize curated pathways) MCODE (Putative protein complexes) GenePro (Protein-Protein interaction cluster visualization) jActiveModules (Search for significant sub-networks) NetworkAnalyzer (Statistical analysis of networks) Agilent Literature Search (Network creation) CyGoose (Gaggle communication)
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Timeseries and BinGO: Aggregate

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Timeseries and BinGO: Analyze

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Timeseries and BinGO

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Timeseries and BinGO

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GOlorize plug-in (Pasteur)

Node placement on the basis of both the connection structure (the edges) and the class structure (GO) A modification of the classic force-directed layout algorithm Beyond GO classes, other class information can be used though attributes (e.g. active modules, complexes)

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GOlorize plug-in interface

Default settings for the class attractive force and separation factor Class-directed network layout
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Example: genetic interaction network

Standard Spring-embedded layout algorithm in Cytoscape

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Example: genetic interaction network

Spring-embedded layout algorithm with GO colour-coding

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Example: genetic interaction network

Final results of the GOlorize layout algorithm in Cytoscape

Garcia et al. Bioinformatics 2007
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Find Network Clusters - MCODE Plugin

Network clusters are highly interconnected sub-networks that may be also partly overlapping Clusters in a protein-protein interaction network have been shown to represent protein complexes and parts of biological pathways Clusters in a protein similarity network represent protein families
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Network Clustering
7000 Yeast interactions among 3000 proteins

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Bader & Hogue, BMC Bioinformatics 2003 4(1):2

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Proteasome 26S
Ribosome

Proteasome 20S

RNA Splicing RNA Pol core

Bader & Hogue, BMC Bioinformatics 2003 4(1):2

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Find Network Motifs - Netmatch plugin

Network motif is a sub-network that occurs significantly more often than by chance alone Input: query and target networks, optional node/edge labels Output: topological query matches as subgraphs of target network Supports: subgraph matching, node/edge labels, label wildcards, approximate paths http://alpha.dmi.unict.it/~ctnyu/netmatch.html
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Finding query sub-networks

Query
Ferro et al. Bioinformatics 2007
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Results

Finding Signaling Pathways

Potential signaling pathways from plasma membrane to nucleus via cytoplasm

NetMatch Results
Signaling pathway example NetMatch query

Ras

MAP Kinase Cascade

TFs

Raf-1
Shortest path between subgraph matches
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Mek MAPK

Nucleus - Growth Control Mitogenesis

Find Active Subnetworks

Active modules are sub-networks that show differential expression over user-specified conditions or time-points
Microarray gene-expression attributes Mass-spectrometry protein abundance

Method
Calculate z-score/node, ZA score/subgraph, correct for random expression data sampling Score over multiple experimental conditions Simulated annealing-based search method is used to find the high scoring networks
Ideker T, Ozier O, Schwikowski B, Siegel AF Human Genetics Bioinformatics. 2002;18 Suppl 1:S233-40

Finding active modules

jActiveModules plug-in
Input: interaction network and p-values for gene expression values over several conditions

Output: significant subnetworks that show differential expression over one or several conditions

Ideker T et al. Science 2001; Bioinformatics 2002

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Cerebral: Cellular location and expression data

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Concluding
Cytoscape is a proven valuable tool for integrative bioinformatics Easily extensible: well suited to answer new biological research questions Analyses can be tedious for biologists; up to bioinformaticians to translate these in simple workflows Therefore: bioinformaticians, integrate into wet-lab research groups!
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Some notes
Plugin lifetime
Maintenance Interoperability

Visualization issues
Standard biologist layouts Fancy visuals

Cytoscape 3.0 aims to solve these issues (amongst others)

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Availability
Cytoscape:
http://cytoscape.org cytoscape-discuss@googlegroups.com cytoscape-helpdesk@googlegroups.com

R2
Available shortly through http://humangeneticsamc.nl Keep yourself posted on http://groups.google.com/group/r2-announce Human Genetics