1. Pathway Analysis
June 13, 2017

2. Cellular context
Pathways provide a more complete picture of the input, output and actual reactions catalyzed by proteins in the cell.
Includes small molecules, type of reaction catalyzed, cellular location
Same proteins can participate in different pathways

3. Pathway Databases Advantages:
More complete & manually curated view of the input, output and actual reactions catalyzed by proteins in the cell.
Includes small molecules, type of reaction catalyzed, cellular location
Same proteins can participate in different pathways
Visualizations
Disadvantages:
Sparse coverage of genome
Different databases disagree on boundaries of pathways.

4. Pathway databases KEGG – Kyoto Encyclopedia of Genes and Genomes
Search, downloads, mapping, enrichment via DAVID
Reactome – EBI
Wikipedia – registered users can contribute
Search, downloads, mapping?
Ingenuity Pathway Analysis (IPA)
$$, won’t use

5. Reactome Hand-curated pathways in human
Rigorous curation standards – every reaction traceable to primary literature.
Automatically project the pathways to non-human species.
Can test for over-representation in DAVID, but maps are not linked directly
Can test of over-representation using Reactome website by submitting a list of genes

6. KEGG Pathways added in a variety of organisms
Some translated to other species but not all
Level of support/annotation for each step is less clear and well defined
Pathway images are static and not linked to other pathways
Easier to use because less complex

7. Accessing pathways Over-representation analysis using DAVID
Will identify pathways over-represented in a gene list and provide genes identified in pathway
Direct links to KEGG pathways
List of over-represented Reactome pathways
Search using a single gene identifier to find pathways that include that gene
Both KEGG and Reactome
Submit list of genes
Reactome only

8. Search of KEGG using TLR4 gene symbol

9. KEGG-TLR4

10. KEGG - notation

11. KEGG-Necrotopsis
Green: human gene names; Pink: disease association

12. KEGG-Necrotopsis
Reference pathway – no hyperlinks

13. KEGG-Necrotopsis
Reference pathway KO – BLUE boxes are hyperlinks to KEGG orthologues

14. KEGG-Necrotopsis
Human pathway– Green boxes are hyperlinks to human gene entries

15. KEGG-Necrotopsis
Disease + Drug – PINK are disease or drug; GREEN/BLUE are gene links

16. Reactome

17. Tlr4 search at Reactome

18. Tlr4 search at Reactome

19. Reactome – TLR4

20. Reactome – Tlr4

21. Reactome - pathways & reactions

22. Reactome – highlights reactions

24. The Pathway Browser - Pathway Diagrams
Catalyst
Inputs
Outputs
Compartment
Reaction node
Regulation
+ve ve
Transition Binding Dissociation Omitted Uncertain
Reactome pathways are represented in diagrams that use a graphical representation standard called Systems Biology Graphical Notation or SBGN. This slide explains most of the diagram elements that are found in a pathway diagram. There is a Diagram Key linked in the top right corner of every diagram. The image at the top of this slide is a typical reaction. At the centre is the reaction node. When this is clicked in the diagram, the entire reaction is selected. Input molecules are connected to the reaction node by plain lines. Output molecules have an arrow head, indicating the direction of the reaction. In this reaction the inputs and outputs are ovals, indicating that they are small molecules. To the left of the reaction node is a rectangle labelled MGLL. The reactangular shape indicates that is it a protein, and the presence of the open circle at the end of the line connecting it to the reaction node indicates that it’s a catalyst for this reaction. If you look at the diagram key on the right, you will see that molecular complexes are represented as blue reactangles with squared-off corners. Sets of functionally-equivalent molecules are shown as blue rectangles that have a double-boundary. Looking at the bottom of the slide, there are several different types of reaction node, representing reaction classes. The default is an open square, which indicates a transition, i.e. a reaction that does not belong to one of the other classes. Binding events are indicated by a filled circle, dissociation by a circle with a double boundary. The square containing two back-slashes indicates an omitted process, i.e an event where the full molecular details are deliberately not shown, typically because they are overly complicated and would take up too much space. A common use of this is to represent the process of transcription and translation following the binding of a transcription factor to a promotor. Rather than show the full, complicated mechanism, which is available elsewhere in Reactome, we would show that the consequence is the appearance of the protein. The square with a question mark inside is used for an uncertain process, where some molecular details are known, but there is a suspicion that there are more molecules involved, or intermediate events that have not been discerned. A common usage of this is in cascading signalling processes. An experiment may show that the overexpression of protein X causes protein Y to be phosphorylated, but the effect may be indirect, if there is doubt then the uncertain class of reaction node would be used to represent this.

25. Pathway enrichment
DAVID – select a gene list and under functional annotation tools, select KEGG pathways

26. KEGG pathways w/ genes
Genes in list

27. Pathway analysis/enrichment - Reactome
Header line must start with #

28. Pathway enrichment – Overview

29. Pathway enrichment - Reactome

30. Today in computer lab
Work with pathway enrichment using DAVID and Reactome
Find a pathway containing one of the genes from your list and compare the information available in Reactome versus KEGG.