1. Pathway Analysis
July 9, 2019

2. 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.

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

4. EBI Reactome Hand-curated pathways in human
Rigorous curation standards – every reaction is traceable to primary literature
Most complete/coverage of publicly available pathways
Automatically project the pathways to non-human species
Can test for over-representation of Reactome pathways in WebGestalt and String-DB
Can also test for over-representation by submitting a list of genes to the Reactome database

5. 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

6. WikiPathways Pathways maintained by and for the scientific community
Co-founded by groups at the Gladstone Institute and Maastricht University
Have a custom graphical pathway editing tool and integrated databases covering major gene, protein and small molecule systems
Enrichment available via WebGestalt only
WikiPathways: a multifaceted pathway database bridging metabolomics to other omics research Nucleic Acids Research, (2017) doi.org/ /nar/gkx1064 PMC

7. Accessing pathways
Over-representation analysis using DAVID, WebGestalt and String-DB
Will identify pathways over-represented in a gene list and provide genes identified in pathway
Direct links to KEGG pathways with genes highlighted
List of over-represented Reactome pathways
Search using a single gene identifier to find pathways that include that gene
KEGG, Reactome and WikiPathways
Submit list of genes
Reactome although the interface is not intuitive

8. Search of KEGG using TLR4 gene symbol

9. KEGG: TLR signaling pathway, reference

10. KEGG - notation

11. KEGG-Necrotopsis
Reference pathway – no hyperlinks

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

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

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

15. Reactome

16. Tlr4 search at Reactome

17. Tlr4 search at Reactome

18. Reactome – Tlr4
Reactome is very interactive and difficult to capture in static images

19. Tlr4 in context of Innate Immunity
Pink outlines represent pathways where Tlr4 is found

20. Tlr4 in context of Programmed Cell Death

21. Interactions between proteins

22. 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.

23. WikiPathways

24. Search for TLR4

25. TLR4 platelet-mediated interactions

26. References for WikiPathways

27. Enrichment Analysis using WebGestalt

28. Pathway Enrichment: WebGestalt
Links to the pathway database

29. KEGG pathway from WebGestalt
Genes from list are shown in red

30. Links to other pathways
Wikipathways from WebGestalt
Links to Reactome pathways, but without genes from list highlighted
Links to WikiPathways will show genes from list (in blue), but can be slow to appear
Issues with image being less responsive
No link to download image; screen capture only way to get it

31. Pathway Enrichment – StringDB
Part of analysis tab
Click on the pathway ID to open up the pathway
The genes from list are not colored in the pathways

32. StringDB – enrichment

33. Pathway analysis recap
Pathways provide context to the PPI and GO enriched gene lists
Pathway building lags behind other types of annotation so coverage can be sparse
Different pathway databases may have different names but cover similar or overlapping protein sets and biological processes

34. Today in computer lab No tutorial
Work with pathway enrichment using WebGestalt and String-DB
Check some of your interacting proteins for presence in the same pathway
Spend some time working through Reactome as it is the most full featured and complete of the publicly available pathways