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

Outline 1. What are biological pathways? 2. Pathway databases 3. Pathway analysis 4. Software: PathVisio 5. Examples 6. Hands on session BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 2

What are biological pathways?

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.” BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 4

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.” BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 5

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.” BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 6

What are biological pathways? Types of molecules : DNA Genomics-25,000 genes RNA Transcriptomics – 100,000 Transcripts PROTEIN Proteomics – 1,000,000 Proteins BIOCHEMICALS Metabolomics – 1,800 Biochemicals (METABOLITES) BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 7

What are biological pathways? Types of interactions : Complex Assembly: Catalysis: Complex Metabolite A Protein 1 Protein 1 Enzyme Protein 2 Protein 2 Metabolite B Transport: Gene regulation: External stimulus Transport in Gene Protein cytoplasm Inhibition Activation TF1 TF2 Cell membrane BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 8

Biological pathway types 1. Metabolic pathways 2. Gene regulation pathways 3. Signal transduction pathways http://www.genome.gov/multimedia/illustrations/Biological_Pathways.pdf BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 9

Metabolic pathways • “… series of chemical reactions occuring within a cell ” • Enzymes calayze the reactions • Example: Glycolysis http://wikipathways.org/index.php/Pathway:WP534 BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 10

Pathway databases

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 ... BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 12

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. BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 13

Pathway databases • WikiPathways (www.wikipathways.org) • Community curated pathway databases What is a wiki? A wiki is an application, typically a web application, which allows collaborative modification, extension, or deletion of its content and structure. BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 14

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 BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 15

WikiPathways BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 16

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

Pathway analysis

Why pathway analysis? Scanned microarrays Image analysis Set of affected Raw pathways Pathway intensities analysis QC Normalization Set of Biological overrepresentated Interpretation GO GO terms Normalized analysis intensities Statistical Set of co-regulated analysis genes Clustering Gene level statistics BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 19

Why pathway analysis? Hmgcr Dgat1 Ldlr Mttp Soat1 Lipc Pltp Lcat BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 20

Why pathway analysis? “A picture is worth a thousand words.” • Intuitive • Puts data into biological context • More efficient than looking up single gene information BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 21

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 BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 22

Pathway analysis methods • Overrepresentation analysis (ORA) • Functional Class Scoring (FCS) • Pathway Topology (PT) Based 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 23

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) BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 24

Pathway analysis methods 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 25

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 BUT ... BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 26

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! BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 27

PathVisio 3.2.0 a tool to edit and analyze biological pathways www.pathvisio.org BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 29

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 BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 30

PathVisio application Toolbar: drawing objects, layout, view, visualization Pathway display area Sidepanel : Drawing, Editing, Backpage, Search, Legend Status bar : Loaded databases, data sets BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 31

PathVisio walkthrough Draw pathways 1 2 Visualize data Load identifier mapping databases Create new pathway Load dataset Share/Upload Pathway statistics 3 Create data visualization WikiPathways Select pathway Visualize data on collection pathway Find regulated Export pathway image pathways with data BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 32

Load identifier mapping databases 0 • 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 Check status bar! BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 33

Identifier mapping • Annotation of data nodes and interactions • Xref → identifier + data source BioSB 2015 course: Biological Network Analysis, 17 & 18 September 2015 34