Pathway Analysis June 13, 2017

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

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.

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

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 www.reactome.org

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 http://www.genome.jp/kegg/pathway.html

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

Search of KEGG using TLR4 gene symbol

KEGG-TLR4

KEGG - notation

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

KEGG-Necrotopsis Reference pathway – no hyperlinks

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

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

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

Reactome http://reactome.org

Tlr4 search at Reactome

Tlr4 search at Reactome

Reactome – TLR4

Reactome – Tlr4

Reactome - pathways & reactions

Reactome – highlights reactions

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.

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

KEGG pathways w/ genes Genes in list

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

Pathway enrichment – Overview

Pathway enrichment - Reactome

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.