Pathway Analysis Susan Steinbusch-Coort, PhD Department of - - PowerPoint PPT Presentation

Pathway Analysis

Susan Steinbusch-Coort, PhD

Department of Bioinformatics-BiGCaT, Maastricht University, The Netherlands

BioSB course: Biological Network Analysis http://tinyurl.com/pl5yreh Amsterdam, 17-18 September 2015 susan.coort@maastrichtuniversity.nl

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Outline

• 1. What are biological pathways?
• 2. Pathway databases
• 3. Pathway analysis
• 4. Software: PathVisio
• 5. Examples
• 6. Hands on session

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What are biological pathways?

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

What are biological pathways?

Definition on Wikipedia (August 2015):

“A biological pathway is a series of interactions among molecules in a cell that leads to a certain product or a change in a cell.”

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

What are biological pathways?

Definition on Wikipedia (August 2015):

“A biological pathway is a series of interactions among molecules in a cell that leads to a certain product or a change in a cell.”

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

What are biological pathways?

Definition on Wikipedia (August 2015):

“A biological pathway is a series of interactions among molecules in a cell that leads to a certain product or a change in a cell.”

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

What are biological pathways?

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DNA RNA PROTEIN Genomics-25,000 genes Transcriptomics – 100,000 Transcripts Proteomics – 1,000,000 Proteins BIOCHEMICALS (METABOLITES) Metabolomics – 1,800 Biochemicals

Types of molecules:

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

What are biological pathways?

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Protein 1 Protein 2 Protein 1 Protein 2 Complex

Cell membrane

External stimulus

Transport in cytoplasm Metabolite A Metabolite B Enzyme Protein Gene

TF1 TF2 Activation Inhibition

Types of interactions:

Complex Assembly: Transport: Catalysis: Gene regulation:

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Biological pathway types

• 1. Metabolic pathways
• 2. Gene regulation

pathways

• 3. Signal transduction

pathways

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http://www.genome.gov/multimedia/illustrations/Biological_Pathways.pdf

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Metabolic pathways

• “… series of chemical

reactions occuring within a cell”

• Enzymes calayze the

reactions

• Example: Glycolysis

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http://wikipathways.org/index.php/Pathway:WP534

Pathway databases

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway databases

• Where can we find biological pathways?

PathGuide:

• List of > 500 pathway and interaction related resources

– http://www.pathguide.org

• Commonly used pathway databases:

– WikiPathways (www.wikipathways.org) – Reactome (www.reactome.org) – KEGG (www.genome.jp/kegg) – BioCyc (www.biocyc.org) – Species specic pathway databases ...

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway databases

• Be aware!
• Only 50% of the human protein coding genes are

present in biological pathways.

• A lot of information hidden in literature and

researchers' minds.

• Detailed functions and mechanisms for many

proteins still unknown.

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway databases

• WikiPathways (www.wikipathways.org)
• Community curated pathway databases

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What is a wiki? A wiki is an application, typically a web application, which allows collaborative modification, extension,

• r deletion of its content and structure.

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway databases

• WikiPathways (www.wikipathways.org)

– everybody can contribute and share pathways – everybody can edit and curate pathways – everybody can use the pathway collection – not just diagrams but fully annotated pathways – interactive pathway viewer – integrated pathway editor – changes can be reverted easily – new findings can be added immediately

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

WikiPathways

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

WikiPathways

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Pathway analysis

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Why pathway analysis?

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Scanned microarrays Raw intensities Normalized intensities Gene level statistics Set of affected pathways Set of

• verrepresentated

GO terms Set of co-regulated genes Biological Interpretation Image analysis QC Normalization Statistical analysis Pathway analysis Clustering GO analysis

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Why pathway analysis?

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Hmgcr Dgat1 Ldlr Mttp Soat1 Lipc Pltp Lcat

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Why pathway analysis?

“A picture is worth a thousand words.”

• Intuitive
• Puts data into biological context
• More efficient than looking up single gene

information

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Why pathway analysis?

• Involvement in pathways

– Group genes, proteins and other biological molecules – Reducing complexity – Several hundred pathways instead of thousands of genes – Analysis on functional level

• Identify active pathways that differ between two

conditions

– Higher explanatory power than a simple list of genes

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway analysis methods

• Overrepresentation analysis (ORA)
• Functional Class Scoring (FCS)
• Pathway Topology (PT) Based

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Khatri, Purvesh, Marina Sirota, and Atul J. Butte. "Ten years of pathway analysis: current approaches and outstanding challenges.“ PLoS computational biology 8.2 (2012): e1002375.

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway analysis methods

• Overrepresentation analysis:

– Define input list through criteria – Count genes in pathway for input and background list – Perform statistical test for over- or under-representation (e.g. hypergeometric test)

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway analysis methods

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• 1. Total number of genes measured in experiment = N
• 2. Set criteria to define total number of “differential expressed genes” = R (p-value<0.05)
• 3. Total number of genes in pathway that are measured in experiment = n
• 4. Number of genes changed in the pathway = r

N = 35 genes R = 9 genes n = 12 genes r = 6 genes Z-Score = 2.34033 Pathway X

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway analysis methods

• What does the Z-Score tell you?

– Z-Score > 1.96:

• Significantly more genes are changed in the pathway compared to

the complete data set

– Z-Score = 0:

• Distribution of changed genes in the pathway is the same as in the

complete data set

– Z-Score < -1.96

• Significantly less genes are changed in the pathway compared to

the complete data set

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BUT ...

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway analysis methods

• Be aware!

– Overrepresentation analysis and functional class scoring DO NOT take pathway topology into account! – Always manually verify the pathways to make the right conclusions!

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

PathVisio 3.2.0

a tool to edit and analyze biological pathways

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www.pathvisio.org

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

PathVisio 3.2.0

• What can you do with PathVisio?

– Draw your pathways – Visualize your data on pathways – Find pathways regulated in your data set

• More functionality through plugins

– Plugin repository with 16 plugins and more coming – Many different plugin developers

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

PathVisio application

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Toolbar: drawing objects, layout, view, visualization Sidepanel: Drawing, Editing, Backpage, Search, Legend Status bar: Loaded databases, data sets Pathway display area

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

PathVisio walkthrough

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Load identifier mapping databases Create new pathway Load dataset Create data visualization Find regulated pathways WikiPathways Export pathway image with data 1 Select pathway collection Draw pathways Visualize data on pathway 2 Visualize data 3 Pathway statistics

Share/Upload

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Load identifier mapping databases

• Identifier mapping datbases from

– Download bridge files from http://bridgedb.org/data/gene_database/ – Gene products (based on Ensembl) – Metabolites (based on HMDB)

• Data -> Select Gene/Metabolite Database

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Check status bar!

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Identifier mapping

• Annotation of data nodes and interactions
• Xref → identifier + data source

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Identifier mapping

How does it work in PathVisio?

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Annotation ID Mapping

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Data visualization

• Load dataset

– Quantitative data – Comma separated file (.csv or .txt) – Identifier column – System code (1-2 letter code) column → datasource (optional)

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Data visualization

• Create visualization

– Basic – one value (e.g. logFC) – Advanced – multiple values (e.g. logFC + p.value)

• Gradient based visualization

– logFC

• Rule based visualization

– p.value

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Data visualization

• Visualize data on

pathway

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Data visualization

• Export high quality pathway image with data for

publications

• Plugins:

– HTMLexport plugin – BioPAX plugin

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BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Pathway statistics

• Download pathway collection from WikiPathways

– http://www.wikipathways.org/index.php/Download_Pathways – 20 different species

• Find regulated pathways

– Default method: Z-Score statistics

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Examples: Pathway Analysis

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Time series data

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JR Tisoncik et al. Microbiology and Molecular Biology Reviews (2012)

Acute-phase response during H5N1 virus infection.

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Tissue expression data

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DGJ Jennen et al. Drug Discovery Today (2010)

Comparison of expression in different cell types.

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Multi-omics data visualization

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I Rubio-Aliaga et al. Physiological Genomics (2011)

Visualization of transcriptomics and metabolomics data together in the pathway.

BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015

Nutritional studies

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M Kutmon et al. Genes & Nutrition (2015)

Cell cycle down- regulation in prostate cancer cells after vitamin D treatment

Hands on session

http://projects.bigcat.unimaas.nl/biosb2015/material/pathway-analysis/

Questions?