- 1 Introduction
- 2 What is Reactome?
- 3 The Reactome Front Page
- 4 The Pathway Browser and Tools
- 4.1 The Pathway Browser
- 4.1.1 The Pathway Hierarchy Panel
- 4.1.2 Pathway Diagrams
- 4.1.3 Navigating Pathway Diagrams
- 4.1.4 Details Panel
- 4.1.5 Context Sensitive Menus in Pathway Diagrams
- 4.1.6 Navigating Disease Pathway Diagrams
- 4.1.7 View Equivalent Pathway In Another Species
- 4.1.8 Molecular Interaction Overlay
- 4.1 The Pathway Browser
- 5 Searching Reactome
- 6 Reactome Tools
- 6.1 Pathway Analysis
- 6.2 Species Comparison
- 6.3 FI Network Tool
- 6.3.1 Overview
- 6.3.2 Download and Launch the Reactome FI plugin
- 6.3.3 Use Reactome Pathways
- 6.3.4 Use the Reactome Functional Interaction (FI) Network
- 6.3.5 Other Features Related to the FI Network
- 7 Download Reactome
- 8 Exporting Data From Reactome
This document is an overview of the Reactome database of biological pathways and processes and its web site. This is not a comprehensive guide, but should provide you with enough information to browse the database and use its principal tools for data analysis. Please read through it and contact us with any comments or questions.
To explain some of the terms used in this User Guide, we have created a Glossary.
What is Reactome?
Reactome is a curated database of pathways and reactions (pathway steps) in human biology. The Reactome definition of a 'reaction' includes many events in biology that are changes in state, such as binding, activation, translocation and degradation, in addition to classical biochemical reactions. Information in the database is authored by expert biologist researchers, maintained by Reactome editorial staff, and extensively cross-referenced to other resources e.g. NCBI, Ensembl, UniProt, UCSC Genome Browser, HapMap, KEGG (Gene and Compound), ChEBI, PubMed and GO. Inferred orthologous reactions are available for over 20 non-human species including mouse, rat, chicken, puffer fish, worm, fly, yeast, rice, Arabidopsis and E.coli.
The Reactome Front Page
This is the normal entry point for most users of Reactome. To view this page, type: http://www.reactome.org into the URL slot on your browser.
This page is divided into six parts:
- The Banner contains a Reactome icon in the banner, which is interactive and when clicked from elsewhere within the website will bring the user back to the homepage.
- The Navigation Bar just below the banner has drop down menus giving access to data and functionality available on the Reactome website.
- Six Tool Buttons on the left-hand side of the page provides shortcut buttons to the most popular analysis tools and downloadable Reactome datasets.
- The Main Text section includes a description of Reactome, the participating research institutions and our funding sources.
- The Twitter feed box on the right-hand side provides up-to the minute Reactome news and a user feedback system.
- The Homepage footer that lists all the webpages accessible throughout the Reactome website.
Most Navigation Bar items are drop down menus. It is found on many pages of the Reactome website. Links from the Navigation Bar include:
- About: Background information such as a description of Reactome and the people involved.
- Content: An overview of the Reactome database, the areas of biology we cover, our plans for content expansion, the structure of our data, and statistics about the current content.
- Documentation: Includes this User Guide, plus technical aspects of Reactome's functionality and data.
- Tools: Tools in addition to those linked as shortcut buttons on the Sidebar. Some of these tools are covered in more detail in the Reactome Tools section.
- Community: Details of Reactome training, publications and representation at conferences and workshops.
- Download: Links to a page where Reactome data and code is available in various formats. See below for more details.
- Contact Us: Opens a form page, which allow you to send your questions or comments to the Reactome helpdesk.
- Search: Type in text (words) or identifiers to locate them in Reactome. For details see Searching Reactome section. Search Examples are listed within the Search text box.
The Six Tool Buttons
The Six Tools Buttons provides easy access to tools, the lower has shortcuts to popular Downloads, Featured tools, and Help. From top to bottom, the features are:
- Browse Pathways: Directly access the Reactome Pathway Browser; see The Pathway Browser and Tools
- Analyze Data: Directly access the most popular Reactome tools; see Reactome Tools for detailed descriptions.
- Reactome FI network: Link to a page describing how to install and use the Reactome Functional Interaction (FI) Network plugin; more details can be found at FI Network Tool
- User Guide: Access this User Guide.
- Downloads: Popular Reactome data downloads.
- Contact Us: Send feedback to Reactome or ask us a question.
The Main Text
This is the text area under the Six Tool Buttons, to the right of the Twitter feed. This area contains the following:
- About Reactome: A one paragraph statement linked to more detailed information.
- Participating Research Institutions: Reactome is a collaboration amongst OICR, NYUMC, CSHL and EBI.
- Funding Agencies: Acknowledging the different funding agencies that support the Reactome project.
The Pathway Browser and Tools
The Pathway Browser is the primary means of viewing and interacting with specific pathways. It includes a search tool, interactive pathway viewer and set of tools for several types of analysis including:
- Pathway over-representation analysis and pathway topology-based analysis
- Comparison of a pathway with its equivalent in another species
- The overlay of user-supplied expression data onto a pathway
- The overlay of protein-protein or protein-compound data from external databases or user-supplied data onto a pathway.
- The display of context-sensitive information from other resources, using widgets provided by the external resources.
The Tool bar on Reactome's Homepage gives access to extended versions of these tools that query across all Reactome pathways (see Reactome Tools).
The Pathway Browser
The Pathway Browser is launched by clicking the Browse Pathways button on the Homepage:
Features of the Pathway Browser are labelled in the diagram below:
- The Features bar across the top of the Pathway Browser has the following items:
- The Reactome logo is a shortcut button that returns you to the homepage.
- Species Selector – Reactome is primarily a database of curated human biological pathways. These human pathways are used to computationally infer equivalent pathways in model organisms (described here). Use the Species Selector to view the pathways inferred for the selected organism. Infectious disease pathways that involve pathogen proteins interacting with human proteins are listed under Homo sapiens.
- Show/Hide toolbar – The pathway browser is divided into 3 main sections. These are 1) the Pathway Hierarchy, on the left, 2) the Details Panel, bottom right, 3) the Diagram Panel, top right. The Show/Hide Toolbar allows you to show or hide these panels.
- Analyse Data – Opens a panel where analyses can be performed
- Quick Tour – click here for a short introduction to Reactome’s key features.
- Diagram Key – this mini key shows the most common objects present in pathway diagrams. Click on the downward-pointing arrow to reveal a more extensive key to pathway diagram features.
- Pathway Hierarchy Panel. The hierarchy of Reactome pathways is listed in the panel on the left side. Similar to the Windows File Manager, sub-pathways can be revealed by clicking on the + symbol to the left of the pathway name. Click on and drag the grey line, separating the Hierarchy panel from the Pathway Diagram Panel, will adjust the width of the Pathway Hierarchy Panel.
- Pathway Diagram Panel. This is where pathway diagrams are displayed, when selected in the Pathway Hierarchy Panel. The Pathway Diagram panel will display a brief guide to the pathway browser until you select a pathway. Top-left of this panel is a navigate/zoom tool. Click on the arrows to move across the diagram. The ‘double arrow” button resizes the pathway diagram to fit the available space. Click on the ‘+’ or ‘-‘ buttons to zoom in and out, respectively. The mouse scroll wheel also zooms.
- Tabbed Details Panel. It gives details of the selected pathway, reaction, complex, set or proteins, when they are selected in the pathway diagram or Pathway Hierarchy panel. This panel can be revealed/hidden using the small yellow triangle on the border with the Pathway Diagram panel. Selecting a location in the pathway diagram with your cursor, and scrolling the wheel will zoom in/out wherever the cursor is located.
The Pathway Hierarchy Panel
This contains a list of Reactome pathway topics, sorted alphabetically. Some topics, such as apoptosis, are too large to represent as a single pathway. Instead they are divided into sub-pathways, which may be further divided into sub-pathways. Most Reactome pathway topics are divided into smaller sub-pathways. Individual steps in a pathway are known as reactions. The organisation of pathways, sub-pathways and reactions is represented on the Pathway Hierarchy panel as a pathway hierarchy. This view functions in a similar manner to the Windows File Manager; sub-pathways are revealed by clicking on the + symbol to the left of the pathway name, and hidden by clicking on the – symbol. The ‘U’, ‘N’ and ‘+’ icons to the left of the pathway name identify whether a pathway is Updated, New or associated with Disease.
Pathways and reactions can be differentiated by a representative symbol to the left of the name, see the image Pathway Hierarchy Symbols below. You may also see a symbol representing 'black-box' reactions, where complete details have been omitted as unnecessary or are not completely determined.
Within a pathway, the order of reactions in the hierarchy from top to bottom usually follows their order in the pathway, so that preceding reactions are above the subsequent reaction, but note that this is not always the case. Reactome has a more formal way of identifying connected reactions called Preceding and Following Events, visible in the Reaction Details (see Details Panel below).
Pathway diagrams represent the steps of a pathway as a series of interconnected molecular events, known in Reactome as 'reactions'. Reactions are the core unit of Reactome's data model. They encapsulate 'changes of state' in biology, such as the familiar biochemical reaction where substrates are converted into products by the action of a catalyst, but also include processes such as transport of molecules from one cellular compartment to another, binding, dissociation, phosphorylation, degradation and more. Cellular compartments are represented as pink boxes - a typical diagram has a box representing the cytosol, bounded by a double-line that represents the plasma membrane. The white background outside this represents the extracellular space. Other organelles are represented as additional labelled boxes within the cytosol. Molecules are placed in the physiologically-correct cellular compartment, or lie on the boundary of a compartment to indicate that they are in the corresponding membrane, e.g. a molecule on the boundary of the cytosol is in the plasma membrane.
A "pop out" diagram key is found in the upper right corner of the webpage.
The diagram key provides a descriptions of the icons used in the diagrams:
Objects on the diagram represent reaction inputs and outputs, and the catalyst if relevant (see A below).
Reactions typically include:
- Inputs, outputs and catalysts, represented as boxes or ovals.
- Green ovals are small molecules or sets of small molecules.
- Green boxes with rounded off corners are individual proteins or sets of proteins or mixed sets.
- Green boxes with square corners are proteins that have no Uniprot accession (or did not at the time the reaction was created).
- Blue boxes are complexes, i.e. proteins and/or small molecules that are bound to each other.
- Green boxes with white inner boxes are sub-pathways.
- Input and output molecules are joined by lines to a central 'reaction node' (surrounded by a green box in Reaction Objects A, below). Clicking this node selects the reaction.
- The outputs of a reaction have an arrowhead on the line connecting them to the reaction node.
- Numbered boxes on the line between an input/output and the reaction node indicate the number of units of this input/output in the reaction (when n >1).
- Reaction input/output molecules are often connected by arrows to molecules that take part in preceding or subsequent reactions (i.e. the preceding/subsequent steps in the pathway).
- Catalysts are connected to the reaction node by a line ending in an open circle.
- Molecules that regulate a reaction are connected to the reaction node by a line ending in an open triangle for positive regulation or a 'T'-shaped head for negative regulation (see B below).
- A white box labelled P on the boundary of an object indicates a phosphorylation event.
- Proteins or small molecules that are also part of a displayed set are connected to the set object by a line of short dashes.
- Sets with overlapping content are connected by a line of long dashes.
- Reactions that represent a disease process use red connecting lines. Objects associated with a disease state are bordered in red.
The reaction node has 5 subtypes, indicating subclasses of reaction (see Reaction Objects C, below):
- Open squares represent a 'transition'
- Filled circles represent 'association', i.e. binding
- Double-circles represent 'dissociation'
- Squares with two slashes represent 'omitted process'. This is used to denote a reaction where the full details have been deliberately omitted. This is most commonly used for events that include specific members of a protein family to illustrate the general behaviour of the larger group. It is used for reactions that occur with no fixed order or stoichiometry, and for degradation where the output is a random set of peptide fragments.
- Squares containing a question mark represent 'uncertain process', where some details of the reaction are known, but the process is thought to be more complex than it is represented. Explanatory details are typically included in the Summation section, described here.
The Pathway Diagram, Hierarchy and Details Panel are interactively connected. Clicking on a pathway or reaction name in the Pathway Hierarchy causes it to be highlighted in dark blue, opens the corresponding pathway diagram in the Pathway Diagram panel and populates the Details panel. If the pathway is a sub-pathway, the name of its parent will be highlighted in pale blue.
Selecting a reaction or pathway in the diagram will highlight it in the Hierarchy, and update the information seen in the Details Panel. If the selected event or molecule is not currently visible the pathway diagram will automatically re-centre on the selected object(s). Selecting a molecule in the diagram will produce details for that molecule in the Details Panel.
Where a subpathway is contained within the diagram that is already displayed, clicking on the sub-pathway name in the hierarchy will cause all the reactions in that pathway to be highlighted in blue.
Highlighted reactions are also visible in the thumbnail diagram and can be used to navigate quickly to the region of interest in the diagram using the thumbnail window, bottom left of the Pathway Diagram. The blue box outline represents the current view. Click and drag blue box will reposition the pathway diagram. To centre the diagram at the point, click outside the blue outline. Moving the cursor out of thumbnail box while dragging will stops the pathway repositioning.
Hovering over a protein, complex or small molecule node shows a tooltip with node's name and type. An information icon to the top left of the node is displayed. Clicking the ‘info’ icon will display a context sensitive menu, providing access to more information about the pathway node.
There is a navigation tool top-left of the Pathway Diagram. With this you can navigate left, right up or down and zoom in or out of the diagram. You can also zoom using the mouse wheel, and click and drag the diagram.
Located at the top right of the Pathway diagram is the Pathway Options button, will display the pathway diagram settings, allowing the user to configure different aspects viewing the pathway diagram and to download the pathway diagram.
Large topics such as apoptosis contain too much information to be displayed as a single pathway or diagram. Where this is the case, the topic is divided into sub-pathways in the pathway hierarchy, and sub-pathway diagrams in the Pathway Diagram Panel. If you select a pathway in the Pathway Hierarchy panel that only has sub-pathways diagrams, the Pathway Diagram displays an overview diagram, containing boxes with green borders, the symbol for a sub-pathway diagram. This symbol indicates that a detailed diagram is available but is not part of the currently displayed Pathway Diagram. The sub-pathway diagram symbol can also be used within a pathway diagram if the details of a sub-pathway are too complicated for inclusion, to indicate that the sub-pathway has it's own, separate diagram.
There are two ways to access a sub-pathway diagram. The simplest is to right-click on the sub-pathway diagram symbol. This produces a menu, select the option 'Go To Pathway'. Alternatively, left-click to select the sub-pathway diagram symbol - the corresponding sub-pathway name will be highlighted on the Pathway Hierarchy panel. In the example shown below, the sub-pathway diagram symbol for 'Extrinsic Pathway for Apoptosis' is selected (boxed in blue) causing the corresponding sub-pathway and it's parent pathway to be highlighted blue on the Pathway tab. The sub-pathway name has not been clicked (that would open the diagram for the subpathway) . Clicking on the sub-pathway name in the hierarchy causes the corresponding pathway diagram to open in the Pathway Diagram panel.
The Details Panel is at the bottom of the Pathway Browser. It gives details of the selected pathway, reaction, complex, set or proteins, when they are selected in the Pathway Diagram or Pathway Hierarchy panel. The height of the Details panel can be adjusted by clicking on and dragging the grey line, separating the Details Panel from the Pathway Diagram, to the intended height.
The different tabs within the “Details" panel, provides alternative displays of graphical and textual information depending on whether a pathway, reaction, or physical entity node is selected:
- Overview: Displays summary information relating to the selected pathway, reaction or entity.
- Molecules: This tab contains a List button which displays details of the molecules associated with the event or object selected in the pathway diagram, including links and accession numbers in various external databases. Molecule information can be downloaded as a csv, tsv, xml or excel file.
- Structures:The content displayed in this tab depends on the type of object or event selected in the pathway diagram. For Reactions it will display equivalent reaction diagrams from the Rhea database when available. For Proteins or pathway objects that represent sets or complexes, corresponding structure information from PDBe is represented if available. For simple molecules, the structures tab represents information from the ChEBI database.
- Expression: Displays condition-specific gene expression data from the Gene Expression Atlas.
- Analysis: This tab displays the results of analyses – see the section covering Reactome Analysis Tools for further details.
- Processes: Events and molecular objects in Reactome are re-used if they can take part in more than one process. You can identify the involvement of a molecule or event in multiple processes by referring to the Processes Tab.
- Download: Allows users to download a variety of compatible with third-party tools
Pathway Details Panel
The Overview tab of the Pathway Details Panel typically includes, the pathway name, the species, a stable identifier, a description of the pathway (summation), the GO biological process term for the pathway, the GO cellular compartment term (if applicable), and PubMed references providing background information and experimental support for the pathway.
If the pathway is not supported directly by human experimental data but has been inferred from another species, this is indicated by the phrase 'This event is deduced on the basis of event(s)' and a link to the supporting pathway. The ‘Computationally inferred to' drop down menu lists species that have been computationally predicted to have the same pathway.
The names of authors and reviewers that contributed to the curation, review and/or updating of the pathway are also provided.
Clicking the ‘+’ or ‘-‘ icon associated with the different displayed fields within the Overview tab will show or hide more information about those fields.
The molecules that participate in a pathway can be displayed and/or downloaded by clicking on the ‘Molecules’ tab of the Pathways Details Panel. The user can select the type(s) of molecules to download, the type of information desired for each molecule type, and the format of the download file.
The 'Expression' tab displays gene expression data derived from transcription profiling of combined individuals and a mixture of RNA from 16 human tissues(Illumina Body Map). The heat map employs a two-dimensional matrix where rows correspond to genes (encoding pathway components) and columns correspond to 16 human tissues, such as adipose, brain, colon, etc. Filled boxes indicate genes expressed. A user will be able to see the Expression profiles from the Expression Atlas for all protein participants in the reaction, or the selected entities. Clicking 'Display levels' will show expression values. A human anatomogram provides a graphical display of the gene expression data as a user clicks on the different tissues or gene expression data presented in the table. Clicking the gender symbols denoting male and female will update the anatomogram with sex-specific gene expression data.
The user can select to display the other Reactome pathways for the selected sub-pathway by clicking the Process tab. Clicking a ‘+’ or ‘-‘ icon next to ‘Involved in pathways’ within the Process tab will show or hide more information about the associated pathway and literature citations.
The Download tab provides options for the user to download different files types for the selected pathways. These files include:
- SBML: An exchange format used by systems biologists for their models.
- SBGN: An exchange format used to represent pathway and network diagrams.
- BioPAX2: An exchange format used by systems biologists for their models.
- BioPAX3: An exchange format used by systems biologists for their models.
- PDF: Text dump of the pathway, organized to look like a research report.
- Word: A document format compatible with Microsoft and other word processor software.
- Protege: A format used for ontology exchange.
SBML, SBGN and BioPAX are exchange formats of interest to bioinformaticians. Word and PDF are familiar document formats, providing you with a convenient "document" of the pathway. Protégé is an extensible, platform-independent environment for creating and editing ontologies and knowledge bases, this download is likely to be of interest to those wishing to extend Reactome functionality.
The complete Reactome textbook of biological processes in PDF or RTF format, the complete set of human reactions in Reactome (in SBML or BioPAX level 2 or 3 format), and a list of human protein-protein interaction pairs are available to download from the Download page, linked to the Menu Bar on the Reactome homepage.
To find out about how Reactome generates SBML, see SBML At Reactome.
Reaction Details Panel
Included in the Overview tab of the Reactions Details Panel is a reaction name, a stable identifier, and a summary of the reaction. Reactions must contain literature references that contain experimental data verifying the reaction, or an 'Inferred From' link if the reaction has been manually inferred from experimental data from model organisms. For pathway details see the previous section.
Other details within the Overview section include:
- Input/Output: Identifies the input/output molecules, sets or complexes for this reaction. Icons to the right of these named items link to further information in Reactome.
- Authors: The expert biologists that contributed materials that allowed this reaction to be created in Reactome.
- Reviewers: The expert biologists that verified the content for this pathway.
- Preceding event(s): A list of events that occur immediately before the event being viewed.
- Following event(s): A list of events that occur immediately after the event being viewed.
- Catalyst (when relevant): The protein or complex that catalyzes the reaction. The Gene Ontology molecular function term that represents the activity of a catalyst or transporter within the reaction will be listed. If the catalyst is a complex, the component, or domain in a simple catalyst, that enables the reaction to occur will be listed.
- Inferred from another species (when relevant): This indicates that event has not been experimentally demonstrated in humans, but has been inferred on the basis of data acquired for another species. The link points to a web page containing the experimental data for the reaction in that species.
- Positively (or Negatively) regulated by: The protein or complex that regulates the reactions.
- References: The research publications that describe the evidence supporting the reaction, each hyperlinked to its PubMed abstract (when applicable).
- Taxon: The species in which the event occurs.
- Computationally inferred to: Links to descriptions of the events in other species that are either confirmed to occur in a very similar way in both species, or have been electronically inferred .
The molecules that participate in a reaction can be displayed and/or downloaded by clicking on the ‘Molecules’ tab of the Reaction Details Panel. The user can select the type(s) of molecules to download, the type of information desired for each molecule type, and the format of the download file.
The 'Expression' tab displays gene expression data derived from the Illumina Body Map for the proteins associated with the selected reaction.
The user can display the other Reactome pathways for the selected reaction by clicking the Process tab. Clicking a ‘+’ or ‘-‘ icon next to ‘Involved in pathways’ within the Process tab will show or hide more information about the associated events and literature citations.
Protein, Small Molecule, Complex and Set Details
The details of any protein, small molecule, complex or set represented in a pathway diagram can be displayed in the Details panel by selecting the object within the diagram. These views are all very similar, an example of protein details is represented below. Clicking a ‘+’ or ‘-‘ icon associated with the different displayed fields within the Detail panel tabs will show or hide more information about the protein, small molecule, complex and set.
The Overview tab displays the following information depending upon whether the selected node is a:
- Link to corresponding entries in other databases: Cross-reference to identifiers used for this molecule in external reference databases, with hyperlinks to the record.
- Cellular compartment: The cellular compartment that contains this molecule, with hyperlink to the corresponding GO term.
- Computationally inferred orthologues: Lists equivalent molecules in other species if these have been inferred to exist. A description of the inference process can be found here.
- Components: If this molecule is part of any complexes, they are listed here
- Produced by: If this molecule is 'created', e.g. becomes incorporated into a complex or degraded, the appropriate reactions are listed.
- Consumed by events: If this molecule is 'consumed', e.g. becomes incorporated into a complex or degraded, the appropriate reactions are listed.
- Post-translational modification: If this molecule exists within Reactome in an alternate, post-translationally modified form, the appropriate molecules are listed.
The ‘Molecules’ tab displays more information and link outs to external bioinformatic resources. The user can select the type(s) of molecules to download, the type of information desired for each molecule type, and the format of the download file.
The ‘Structures’ tab will displays 3D structural data and citations from PDB. The most relevant (best) crystal structure for the selected node will be shown. Clicking the "All structures..." will display other associated crystal structure. Users can also explore additional crystal structure infromation at PDB by clicking the different icons:
- Book: A paper describing the PDB entry.
- Person/Multi-Cladogram: The taxon or source of the biomolecule.
- Circular plasmid/Multi: The expermental technique used to create the crystal.
- NMR/X-RAY: The technique used to determine the 3D structure.
- Protein Domain: The protein sequence and domain information associated with protein.
- DNA helix: Nucleic acids (DNA, RNA) are associated (green-highlighted) or not associated (grey-highlighted) with the crystal structure.
- Benzene ring: Ligand is associated (green-highlighted) or not associated (grey-highlighted) with the crystal structure.
The 'Expression' tab displays gene expression data derived from transcription profiling of individual and mixture of 16 human tissues RNA (Illumina Body Map). The heat map employs a two-dimensional matrix where rows correspond to genes (encoding pathway components) and columns correspond to 16 human tissues, such as adipose, brain, colon, etc. Filled boxes indicate genes expressed. Click 'Display levels' to show expression. A human anatomogram provides a graphical display of the gene expression data as a user clicks on the different tissues or gene expression data presented in the table. Clicking the gender symbols denoting male and female will update the anatomogram with sex-specific gene expression data.
The ‘Process’ tab shows the Reactome events associated with the selected node. Clicking the ‘+’ or ‘-‘ icon next to ‘Involved in pathways’ will show or hide more information about the associated events and literature citations. "Is present in sets" displays information about the Reactome sets that the selected node participates in.
Context Sensitive Menus in Pathway Diagrams
Within pathway diagrams, hovering on a node will display an information icon, which when clicked will display a context sensitive menu. Alternatively, right-clicking on the box representing a molecule, complex, set or reaction presents the user with a menu or list of features dependent on the nature of the item selected. Note that unavailable options do not appear, so very few items will have the full range of options.
A context sensitive menu is engaged when a user clicks on the the pathway diagram background (space between nodes and reactions). This menu allows the user to explore additional features of the pathway diagram. Some of these features from this menu are also accessible from the Pathway option tool button that is accessible from the top-right corner of the pathway diagram.
Right-click Menu Items associated with Pathway Nodes
- Other Pathways
- Lists other pathways that include the selected item as a participant. Clicking on any of the pathway names will display that pathway in the Pathway Diagram Panel.
- Display Interactors
- Surrounds the selected diagram item with a set of boxes representing protein-protein or protein-small molecule interactors. For more information see Molecular Interaction Overlay. N.B. Interactors can only be displayed for individual proteins, not for complexes or molecule sets.
- Hide Interactors
- Removes interactors from the selected diagram item, unless it is an interactor of another item in the display. For more information see Molecular Interaction Overlay
- Export Interactors
- Exports all interactors of the selected diagram item from the pre-selected source interaction database and displays them in a new browser tab/window. Interaction data is displayed in PSI-MITAB format. For more information see Molecular Interaction Overlay
- Participating Molecules
- Lists the component molecules for the selected item, e.g. lists the proteins that make up a complex. Clicking on any molecule in that list will open a new window with details.
- Display Participating Molecules
- This popup menu item is displayed following Species Comparison or Expression Analysis. It causes the display of a grid of cells that represent the components of the set or complex. See the relevant sections of the User Guide for more details.
- Go To Pathway
- Associated with symbol for sub-pathway diagrams, selecting this from the popup menu causes the sub-pathway diagram to be displayed. See Sub-pathway Diagrams
Right-click Menu Items associated with Pathway Diagram
This menu is engaged when a user clicks on the the pathway diagram background (space between nodes and reactions). This menu allows the user to explore additional features of the pathway diagram.
- Search Diagram (ctrl-f)
- A user can search for a word or phrase within the nodes and reactions of the pathway diagram. The search tab appears with by pressing the 'ctrl-f' key combination. See Search Pathway Diagram
- Snapshot Current View
- Permits the user to download the current pathway diagram view, including overlays, as a PNG image file that is opened in a new tab.
- Download Diagram
- Allows the user to download the entire pathway diagram, including overlays, as a PNG image file that is opened in a new tab.
- Interaction Overlay Options...
- Opens a pop-up box for configuration of the interaction overlay. For more information see Molecular Interaction Overlay
- Zoom In
- Zoom in within the pathway diagram.
- Zoom Out
- Zoom out within the pathway diagram.
- Center Diagram Here
- In combination with the functions of the mouse scroll wheel, the user can zoom in/out within the pathway diagram, relative to the position of the mouse cursor.
Pathway Options Menu
This button, located on the top-right of the Pathway diagram, opens a control panel that has several functions:
- Search Diagram (ctrl-f).
- Search the diagram nodes and reactions for a word or phrase. The search tab appears with by pressing the 'ctrl-f' key combination. See Search Pathway Diagram for more information.
- Snapshot Current View.
- Take a screenshot of the current pathway diagram view, which will be opened in a new window.
- Download Diagram.
- Will open the entire pathway diagram in a new window, ready for download.
- Interaction Overlay Options…
- Allows configuration of Molecular Interactions onto the pathway diagram.
Biological processes are captured in Reactome by identifying the molecules (DNA, RNA, protein, small molecules) involved in them and describing the details of their interactions. From this molecular viewpoint, human disease pathways have three mechanistic causes:
- the inclusion of microbially-expressed proteins
- altered functions of human proteins
- changed expression levels of otherwise functionally normal human proteins
For some Reactome disease pathways, such as "Signaling by EGFR in cancer" it is helpful to describe the effects of disease related reactions and entities on the normal human pathway. In this case, disease related events and normal events are grouped together in the pathway hierarchy and the disease events (reactions and associated molecular entities) are overlaid on top of the diagram of the normal human pathway that they affect. This “merged" diagram can be viewed by clicking on the "parent" disease pathway. Disease related entities and reactions are outlined in red whereas the normal events and entities are “greyed out”. In the event hierarchy, the parent pathway contains both the normal pathway (Signaling by EGFR) and a pathway containing uniquely the disease associated reactions (Signaling by constitutively active EGFR).
Clicking on the “normal" subpathway hides the diseased entities and reactions entirely from the diagram allowing the user to browse just the normal events.
Clicking on the “disease" subpathway shows the merged (disease/normal) diagram again. The disease reaction nodes are highlighted with green boxes. Disease related pathways, reactions, and entities may be flagged, in the details section, with the most relevant EBI Human disease ontology term.
- Note: It is not possible to link to the Human disease ontology website directly from the disease tagged entity/event. This feature will appear in a future release.
Some disease pathways in Reactome have not been grouped, in the hierarchy, with a separately annotated "normal" pathway and diagram. These include infectious disease pathways (and the Amyloids pathway) in which the majority of annotated reactions involve pathogen (abnormal) proteins only and/or involve interactions among pathogen (abnormal) and human proteins. These disease pathways have each been placed in a single diagram. Red lines are used to emphasize disease events. Reactions involved in disease or disease progression are highlighted in red as are any entities that are disease-associated or derived from another species (pathogen). Host/human or normal entities are black. Complexes that contain both host and disease associated/pathogen derived entities are colored red.
View Equivalent Pathway In Another Species
Reactome is human-centric and aims to represent human biology. Pathways in other species are electronically inferred from curated human pathways - a description of the inference process can be found here.
To view the predicted conservation of the displayed pathway in another species, use the "Switch Species" drop-down menu at the top of the Pathway Browser. Selecting causes the pathway diagram to re-draw (a revolving arrow icon on the Pathway Hierarchy panel indicates the diagram is re-drawing). The layout of the diagram is preserved, but any reactions that were not inferred will be absent. The event hierarchy is also updated to display the events present in the selected species.
Note however that when the species Gallus gallus. is selected (this species has both manually curated and electronically inferred pathways in Reactome), it is currently only possible to view the manually curated Gallus pathways. To view the electronically inferred Gallus pathways, you must go to a pathway of interest in human and select the electronically inferred Gallus event in the details section. The inferred Gallus pathway diagram will then be displayed, but the manually curated pathways for Gallus will be listed in the event hierarchy. To navigate within these inferred Gallus pathway diagrams, you can select individual reaction nodes or reaction participants and see their descriptions in the details panel.
A more sophisticated method of comparing pathways between species is available as the Species Comparison tool.
Molecular Interaction Overlay
Molecular Interaction (MI) overlay allows protein-protein or protein-compound interactions to be overlaid (superimposed) onto the pathway diagram. The source depends on the currently selected interaction database. The default is IntAct, other sources of interaction data (protein-protein and protein-compound) including a user-supplied list can be selected using the ‘Interactor Overlay Options…’ feature of the Pathway Options button in the top-right corner of the Pathway diagram.
A maximum of 10 interactors are displayed as a ring of blue-bordered boxes connected by blue lines to the selected protein. A white box superimposed onto the selected protein displays the number of interactors up to a value of 50, if more than 50 are identified 50+ will be displayed.
Several items can be sequentially selected for interactor display. If the same interactor is connected to more than one item it is re-used, i.e. connected to all the selected items in the diagram.
- Hovering the mouse pointer over a protein interactor produces a pop-up containing the name and identifier of the protein.
- Hovering over a chemical interactor displays the formula and name of the chemical.
- Clicking on a protein interactor opens the Uniprot entry for that protein in a new window.
- Clicking on a chemical interactor opens up the ChEBI entry for that chemical in a new window.
- Clicking on the line that connects the interactor to the pathway item opens a new window containing details of the interaction at the source database (see example below) ** Please note that link to the source database is not currently working for Internet Explorer 8**.
Details of up to 50 interactors for every protein in the pathway diagram can be viewed as a table accessed via the Analyze, Update & Annotate button. An extended version of this table with no limit to the number of interactors per protein can be exported. See Analyze, Update & Annotate button for details.
While interactors are displayed, right clicking on the selected protein produces two new options, Hide Interactors which removes them, and Export Interactors. Interactions are exported in PSI-MITAB format.
MI Overlay tab
This tab contains the following features:
- Interaction database - a drop-down list allows selection of the source of interactors. This is automatically populated by querying the PSICQUIC Registry. If a new database is selected while interactors are displayed on the pathway diagram, the proteins represented by those items will be used as queries in the new database and the display will automatically update. If the table of interactions is displayed, it will automatically update to list the interactions found in the new database.
- Upload a file - allows a user supplied list to be used as the source of interactors. Data must be in PSI-MITAB format though the only columns that need to be filled in are the accession number, gene name and confidence score columns (confidence score is only necessary if you want to use the color interactions feature). If the upload is successful, the label you submit when prompted will appear on the 'Interaction Database' drop down and can be selected as a source of interactions. A data set submitted in this manner will persist for the user session.
- Clear overlay - removes all interactors from the pathway diagram.
- Submit a New PSICQUIC service - can be used to add a new source interactions database. The URL field should contain the URL of the PSICQUIC service (REST interface). A service added this way will appear on the interaction database drop down for a period of three days, assuming you connect from the same computer.
- Set Confidence Level Threshold - this allows the user to set a confidence threshold used for colouring interactors. The default threshold is 0.5. Interactions with a confidence level below this threshold will be coloured according to the color set as the 'Below' colour, interactions with confidence scores equal to or above the threshold will be coloured with the 'Above' colour.
- Colour button - activates colouring of the pathway diagram - interactions will be coloured only if a confidence score is available at the source database. The colours used can be changed by clicking on the coloured squares for 'Above' and 'Below'. A dialog allows selection of an alternative colour. Click 'Apply' to update the colours displayed.
- Colouring Off button - removes colouring.
The colours used for interaction colouring can be changed by clicking on the coloured squares for 'Above' and 'Below'. A dialog allows selection of an alternative colour, click 'Apply' to update the colours displayed.
- Display/Hide Table of all interactors for pathway - this button switches on/off the table of pathway item-interactor pairs.
- Clicking on the blue 'toggle' squares within this table will cause the pathway diagram to centre on the protein represented in the first column and display its interactors.
- All entries (rows in the table) for that pathway protein will be shaded a pale blue when interactors are displayed. Clicking the toggle for a particular pathway protein a second time will hide the interactors and its table entries will be coloured white.
- Clicking on a pathway protein ID (listed in the second column of the table) will re-centre the pathway diagram on that protein.
- Export all interactors for pathway - exports a full list of interactors for each protein on the pathway diagram, in PSI-MITAB format, as a new window.
Search Pathway Diagram
The Search Diagram (found in diagram options menu) permits the selection of an entity matching the search term and centers the diagram if it is not visible. The "Select All" feature allows for navigation of all entities matching the search query. "Next" and "Back" allows the user to cycle through the diagram search results.
A number of options are available for searching Reactome, depending on your requirements. The "simple search" will be quite sufficient for most users. If you need to make more complex, logical, queries, then you will need to use the Advanced search. The simple and advanced search features are accessible from the top right of the Reactome home page banner and under “Tools” on the Reactome homepage banner, visible from all pages except the Pathway Browser. respectively.
The simple text search tool is located top right of the homepage banner, visible from all pages except the Pathway Browser. Type the word, identifier or accession number and then click on the "Search" button. You will be presented with a results page similar to this:
Results are displayed in a context dependant mannner depending on your query. Results are ordered into the categories: Proteins, Sets, Complexes, Chemical Compounds, Reactions, Pathways, and other entities, if your query matches Reactome annotations. Clicking on the title (URL) will redirect you to go to the corresponding Reactome web page with additional information about your search query. Some results will have descriptive text details, whereas others might link directly to the Pathway Browser. Your search terms will be highlighted if they appear within the title or descriptive text.
By default, the results are displayed for human species and entries that do not belong to a particular species. The latter is relevant to searches involving small molecules.
At the top right of the results page is a set of tick boxes, to filter and refine your search. Selecting one or more of the selector boxes will automatically restrict your search and refresh the search results page. The clicking one or more of the selector boxes will preform the following:
- Species: restrict the search to a particular species.
- Types: display only information relating to Protein, Set, Complex, Chemical Compound, Reaction, Pathway or OtherEntity.
- Compartments: filter your results for a particular cellular compartment(s).
- Reaction types: display results for specific types of reactions.
- Search properties: clustered search.
By selecting a protein link from within the initial search results, the following page will be displayed describing the protein annotations:
By selecting a RNA sequence link, the following page will be displayed describing the RNA annotations:
By selecting a Set link, the following page will be displayed describing the component set annotations:
By selecting a Complex link, the following page will be displayed describing the complex annotations:
By selecting a Chemical Compound link, the following page will be displayed describing the chemical, small molecule and drug annotations:
Within each of these search results pages there are a number of link outs to further information within Reactome and other third party bioinformatics resources. By selecting a Reaction or Pathway link, the user will be redirected to the Pathway Browser displaying the selected pathway diagram.
The Advanced Search form can be accessed under Tools on the Reactome homepage Menu Bar, visible from all pages except the Pathway Browser. This search method supports full Lucene QueryParser syntax including:
- Quotation marks for exact searches and brackets for grouping terms together
- Boolean operators: 'AND', 'OR', 'NOT', '+' and '-'
- Wildcard operators: '?' and '*'
- Proximity matching: "raf map"~4 will search for raf and map within 4 words from each other
There is also the possibility of specifying the field you want to search in with, such as: dbId, stId, name, type, species, synonyms, summation, compartmentName, compartmentAccession, goBiologicalProcessName, goBiologicalProcessAccession, goCellularComponentName, goCellularComponentAccession, goMolecularFunctionName, goMolecularFunctionAccession, literatureReferenceTitle, literatureReferenceAuthor, literatureReferencePubMedId, literatureReferenceIsbn, crossReferences, referenceCrossReferences, referenceName, referenceSynonyms, referenceIdentifier, referenceOtherIdentifier, referenceGeneNames. The syntax for this field-specific query is fieldname:searchterm.
To make full use of the Advanced Search we recommended that you read the document Data Model (available under Documents from the Menu Bar on the Reactome homepage) and refer to the Database Schema (linked to the Data Model document and under Contents from the Menu Bar).
This example provided in the Advanced search form page will identify Reactome annotations, across all species, with names that include the words "raf" and "map" or "PTEN S170N", "apoptosis" or the stable identifier "REACT_12858.1". By selecting different "Filtering Parameters", the query can be refined to display specific information.
Small Molecule Search Tool
This is accessed via the Menu bar on the Reactome homepage, select Tools, Small molecule Search. This will open the Small molecule search page where you can specify the small molecule by name or by structure. Structures can be hand-drawn, or pasted as a SMILES string. Options include search for molecules that contain this structure or those that resemble this structure. Click on the Search button to start the search. The tool will search the ChEBI database. A list of matching compounds is returned, with links to the appropriate Reactome pathways.
Author/Reviewer Search Tool
Reactome depends on collaboration between our curation team and outside experts to assemble and peer-review its pathway modules. The integration of ORCID within Reactome enables us to meet a key challenge with authoring, curating and reviewing biological information by incentivizing and crediting the external experts that contribute their expertise and time to the Reactome curation process. More information is available at ORCID http://orcid.org and Reactome.
This is accessed via the Menu bar on the Reactome homepage, select Tools, Author/Reviewer Search. This will open a web page entitled "Query your pathway contributions in Reactome" This tool allows users to query the Reactome database for their pathway contributions, and download their contributions as a citation for importation into ORCID.
This tool allows searching for author/reviewer by name or email address, which returns the contributor’s ORCID ID, if available, and links to all pathways the person has either authored or reviewed.
The tool also offers links for creating or reporting ORCID IDs and functionality to export author contributions in BibTeX format suitable for importing to the person’s ORCID account.
The Pathway Analysis Portal combines a number of analysis and visualization tools to permit the interpretation of user-supplied experimental data sets. This portal is launched by pressing the "Analysis-Magnifying Glass" Button in the Feature Bar of the Pathway Browser, the "Analyze Data" button on the homepage, or the "Analyze Data" link from the "Tools' drop-down menu.
The portal opens a submission form, where you can select the analysis you want to perform, paste in or browse to a file containing your data, or use an example data set. The submission process recognizes many types of identifiers. As part of the pre-analysis, they are mapped to Reactome molecules. The ideal identifiers to use are UniProt IDs for proteins, ChEBI IDs for small molecules, and either HGNC gene symbols or ENSEMBL IDs for DNA/RNA molecules, as these are our main external reference sources for proteins and small molecules. Many other identifiers are recognized and mapped to appropriate Reactome molecules. Accepted identifiers include HUGO gene symbols, GenBank/EMBL/DDBJ, RefPep, RefSeq, EntrezGene, MIM, InterPro, EnsEMBL protein, EnsEMBL gene, EnsEMBL transcript, and some Affymetrix and Agilent probe IDs.
Uniprot isoforms may be specified using the format P12345-2. If the –n suffix is omitted this canonical form and all isoforms of it will be matched. Mixed identifier lists may be used. Identifiers must be one per line. Protein-specific identifiers will typically map to protein entities, while gene-specific identifiers will map to the gene, transcript and derived proteins. If desired results can be filtered to show protein-specific or gene/transcript-specific results, details below.
The initial outputs from the analysis is dependent on the dataset uploaded, i.e. a gene list, non-human vs human data, or an expression dataset.
Gene list Dataset
If the dataset is an user-supplied set of human gene or protein identifiers, the analysis portal performs an enrichment test to determine whether any Reactome pathways are enriched in the submitted data, i.e. it answers the question 'does the list represent the proteins within a specific pathway more than would be expected if the set were random?'. A one-tailed Fisher's exact test is used to calculate the probability. The p-values are corrected for multiple testing that arises from evaluating the submitted list of identifiers against every pathway.
The initial results of the overrepresentation analysis are displayed as a table in the "Analysis results" tab. Analysis results are shown in the Analysis tab, within the Details Panel. All Reactome pathways are shown, in blocks of 20 pathways, ranked by the FDR value obtained from over-representation analysis. In addition, the number of molecules matched/total number of molecules and FDR values are added to the right side of pathway names in the Pathway Hierarchy Panel. Reactions that contain any of the matching molecules are boxed in orange. When you click on the name of a pathway in the Analysis tab, the Pathway Hierarchy will expand to show it and its name will be highlighted in dark blue. If it has subpathways, you can use the plus buttons to the left of the pathway name to reveal subpathways and reactions. The columns represent:
- Pathway name: Click the name to open the pathway.
- Entities found: the number of molecules of the type selected with Results Type that are common between the sumbitted data set and the pathway named in column 1. Click on this number to display the matched submitted identifiers and their mapping to Reactome molecules.
- Entities total: The total number of molecules of the type selected with Results Type within the pathway named in column 1.
- Entities ratio: The ratio of entities from this pathway that are molecules of the type selected with Results Type Vs. all entities of the type selected with Results Type.
- Entities pvalue: The result of the statistical test for over-representation, for molecules of the results type selected.
- Entities FDR: False discovery rate. Corrected over-representation probability.
- Reactions found: The number of reactions in the pathway that are represented by at least one molecule in the submitted data set, for the molecule type selected with Results Type.
- Reactions Total: The number of reactions in the pathway that contain molecules of the type selected with Results Type.
- Reactions ratio: The ratio of reactions from this pathway that contain molecules of the type selected with Results Type Vs. all Reactome reactions that contain molecules of the type selected with Results Type.
- Species Name: The species name.
Clicking on any of the Pathway Names in the "Analysis results" table launches the Pathway Browser in a new browser tab and displays the relevant pathway diagram (see example below).
Nodes in the pathway diagram are yellow-coloured when a gene within your list matches a protein with the pathway diagram
- Protein Nodes - coloured yellow. Hovering the mouse pointer over a protein node displays the submitted identifier and the expression value.
- Pathway Nodes – coloured yellow in horizontal segments with each segment representing a protein in the pathway
- Set and Complex Nodes - coloured yellow in horizontal segments with each segment representing a component in the set/complex that is a protein. Right clicking on the set/complex icon and select 'Display Participating Molecules'. A popup is displayed representing the set/complex as a table with the component name, expression identifier, and the expression level.
- White nodes represents proteins or small molecules with not matches in the uploaded data set.
Hovering the mouse pointer over a cell displays the name of the protein. Clicking on the cell displays details of the protein in the Details pane. The grid can be closed by clicking the 'x' in the top right corner.
By default, all molecules (protein, small molecules, genes, transcripts) are used for overrepresentation analysis. Subtypes of molecule can be selected from a drop-down list located top-left of the results table. Selecting of these subsets will re-display results that consider only the selected molecular subtype.
Dataset from a Model Organism
By default, all non-human identifiers are converted to their human equivalents. If you want to use non-human identifiers to search a computationally-inferred non-human Reactome pathway, uncheck the box. You may also prefer to uncheck this box if your query consists of a mixture of human and microbial identifiers, representing an infection.
"Project to Human" Pathway Analysis
The results are ready to view when analysis results appear (or are updated if already present) in the Pathway Hierarchy. Clicking on a pathway name launches the Pathway Browser and displays the relevant Pathway Diagram.
The results page is very similar to that seen following submission of a simple one-column list of identifiers.
"Computationally Inferred Non-Human" Pathway Analysis
The results are ready to view when analysis results appear (or are updated if already present) in the Pathway Hierarchy. Clicking on a pathway name launches the Pathway Browser and displays the relevant Pathway Diagram.
The results page is very similar to that seen following submission of a simple one-column list of identifiers.
If you submit data in a format that includes columns of numeric values, following a first column of protein, compound or other suitable identifiers such as probe IDs, the analysis tool will interpret your data as expression data. The numeric values are used to colour objects in pathway diagrams. This view was created for microarray data, but any dataset that consists of a list of identifiers with associated numeric values can be used, e.g. quantitative proteomics, GWAS scores.
The tool is launched using the Analyse data button in the Pathway Browser header bar. Either paste your data into the submission form or browse to a saved file (or select an example file).
The results page is very similar to that seen following submission of a simple one-column list of identifiers, with extra columns in the Analysis details following column 9. These extra columns represent the submitted expression values. Clicking on a pathway name launches the Pathway Browser and displays the relevant Pathway Diagram.
Nodes in the pathway diagram are coloured according to the user-submitted numerical values. The yellow-blue colour range is used to distinguish the expression values, employing a linear spectrum from yellow for the highest values to dark blue for the lowest values. The scale automatically adjusts to fit the numerical range represented in the dataset.
- Protein Nodes - yellow or blue colour depending on expression value. Hovering the mouse pointer over a protein node displays the submitted identifier and the expression value.
- Pathway Nodes – coloured in horizontal segments (from lowest to highest expression level) with each segment representing a protein in the pathway
- Set/Complex Nodes - coloured in horizontal segments (from lowest to highest expression level) with each segment representing a component in the set/complex that is a protein. The value associated with each component of a complex can be viewed; right click on the complex and select 'Display Participating Molecules'. A popup is displayed representing the complex as a table with the component name, expression identifier, and the expression level.
- White nodes represents proteins or small molecules with no numerical data are coloured white.
Hovering the mouse pointer over a cell displays the name of the protein. Clicking on the cell displays details of the protein in the Details pane. The grid can be closed by clicking the 'x' in the top right corner.
The Experiment Browser toolbar (orange bar with pale blue arrows, at the bottom of the Pathway Diagram) allows you to step through the columns of your data, e.g. time-points or disease progression. Move between them by pressing the arrow buttons. The header of the data column (if present) is displayed between the arrows. The pathway diagram will re-colour to reflect the new values.
Reactome uses manually-curated human pathways to electronically 'infer' their equivalents in 19 other species. A full description of the inference process can be found here. The Species Comparison tool allows you to compare human pathways with these predicted pathways, to see what is common to both or perhaps missing in the model organism.
Species Comparison is launched using a button on the sidebar, on the left side of the homepage. On the resulting page is a selection tool that reveals a drop-down list of species.
Choose one and click the Apply button. It may take some minutes before the results appear. The results page will look something like this:
The colour of reaction objects indicates the result of the comparison:
- Yellow indicates that the protein has an inferred equivalent in the comparison species.
- Blue indicates that no equivalent was identified. This protein may not exist in the comparison species.
- White indicates that inference was not possible. This is always the case for small molecules, DNA and other objects that have no UniProt entry (or did not at the time the pathway was constructed).
- Objects with bands of colour represent complexes or sets containing more than one molecule. The bands of colour reflect the inference success for the molecules within the complex/set.
To view species comparison results for a complex or set right click it and select the option Display Participating Molecules. This reveals a table representing all the proteins involved in the complex/set. Each square in the grid represents one component of the complex/set, coloured as described above.
FI Network Tool
Reactome Cytoscape Plugin is designed to find pathways and network patterns related to cancer and other types of diseases. This plugin accesses the Reactome pathways stored in the database, help you to do pathway enrichment analysis for a set of genes, visualize hit pathways using manually laid-out pathway diagrams directly in Cytoscape, and investigate functional relationships among genes in hit pathways. The plugin can also access the Reactome Functional Interaction (FI) network, a highly reliable, manually curated pathway-based protein functional interaction network covering over 50% of human proteins, and allows you to construct a FI sub-network based on a set of genes, query the FI data source for the underlying evidence for the interaction, build and analyze network modules of highly-interacting groups of genes, perform functional enrichment analysis to annotate the modules, expand the network by finding genes related to the experimental data set, display pathway diagrams, and overlay with a variety of information sources such as cancer gene index annotations. For an example how we use Reactome FIs for cancer data analysis, please see our publication: A human functional protein interaction network and its application to cancer data analysis.
Download and Launch the Reactome FI plugin
Reactome FI Cytoscape Plugin 4 needs Cytoscape 3.0 or above. If you have not installed Cytoscape 3.0 or above, please download it from Cytoscape's web site: http://www.cytoscape.org. After launching Cytoscape, use menu "Apps/App Manager" to open the "App Manager" dialog, and search for "ReactomeFIPlugIn". You should see the Reactome FI plugin listed in the middle panel (See figure below. You may see a different version number). Choose the found plugin, and then click the "Install" button at the bottom of the dialog. Follow the procedures to finish the installation.
Note: The Java Web Start version supports Cytoscape 2.7 with Reactome FI Plugin 3.0 or below (See Download and Launch FI Plugin 3.0). To use our Reactome pathway features, please use plugin 4 with Cytoscape 3.0 by installing a desktop version of Cytoscape 3.0 or above.
Use Reactome Pathways
Showing Reactome pathways directly in Cytoscape is a new feature in the Reactome FI plugin version 4.0. Using this feature, you can load pathways in the Reactome database into Cytoscape, visualize Reactome pathways in either the native pathway diagram view or the FI network view, do pathway enrichment analysis for a set of genes, and check genes from your list in hit pathways.
Explore Reactome Pathways
- Load Reactome pathways: Use menu "Apps/Reactome FI/Reactome Pathways" to load pathways into Cytoscape. The loaded pathways are organized in a hierarchical way as in the Reactome web application (http://www.reactome.org/PathwayBrowser/), and listed in the left side "Control Panel" as a tab called "Reactome".
- View pathways in Reactome: After selecting a pathway in the pathway hierarchy, you can choose "View in Reactome" from the popup menu (right click in Windows or Control-click in Macs to get the popup menu)
Note: The ancestor pathways (container pathways) for a selected pathway are displayed in the middle panel, "Selected Event Branch", in the Reactome tab. You can click an ancestor pathway in this middle panel to view the clicked pathway's location in the original pathway hierarchical tree. However, the ancestor pathway will not be selected in the original tree. This is a designed behavior to keep the selection in the original tree.
- Search pathways: Choose "Search" in the popup menu to bring up the search dialog. The found pathway(s) will be highlighted in blue in the pathway tree.
Note: Search will be against all loaded pathways, not limited to the selected pathway and its contained sub-pathways.
- Open pathway diagram: Pathways in Reactome are organized in a hierarchical way. Not all pathways have their own pathway diagrams. A smaller pathway (called sub-pathway) may be drawn in a bigger pathway, which has its own pathway diagram. Most of top-level pathways (called modules or super pathways) are used to organize related pathways (e.g. Disease, Signaling Transduction), and therefore contain only rectangle boxes representing canonical pathways.
- Show Diagram: If a selected pathway has its own pathway diagram, you can choose "Show Diagram" in the popup menu to open its pathway diagram into the central Cytoscape desktop.
- View in Diagram: If a selected pathway is laid-out as a sub-pathway in a bigger one, you can choose "View in Diagram" in the popup menu to view its drawing in its container pathway. Reactions contained by the selected pathway will be highlighted in blue after the diagram is opened. For example, see pathway "G1/S DNA Damage Checkpoints" opened in pathway "Cell Cycle Checkpoints" below:
- Search diagram: Objects displayed in a pathway diagram can be searched using "Search Diagram" from the popup menu (Right click in Windows or Control click in Macs without selecting any object in the pathway diagram to get the popup menu). The found objects will be selected and highlighted in blue.
Note: reactions will not be searched in the diagram. Use the search feature in the pathway tree to search for reactions.
- Export diagram: Displayed diagram can be exported as a PDF, JPG or PNG file. Use "Export Diagram" from the popup menu to export the displayed diagram.
- View in Reactome: Select an object, and then right-click (or control click) to get the popup menu. Choose "View in Reactome" to view the selected object in the Reactome web application.
- List Genes: Genes contained by a complex or protein set, or a gene related by a displayed protein can be viewed by using a menu item "List Genes" after selecting an object. For example, the following dialog shows genes contained by complex hBUBR1:hBUB3:MAD2*:CDC20. Clicking a gene symbol will bring you to the web page for that gene in the GeneCard web site.
Display Reactome Pathways in the FI Network View
- Display pathway in the FI network view: A Reactome pathway can be converted into a functional interaction network using the method we have established (see A human functional protein interaction network and its application to cancer dat analysis). Use "Convert as FI Network" in the popup menu brought up by right-clicking (Windows) or control-clicking (Macs) an empty area without any selection in the pathway diagram panel. The original pathway diagram will be moved to the bottom-left corner, and a new FI network will be generated based on the original pathway diagram, which will be displayed in a new network panel.
Note: sub-pathways contained by the displayed pathway will be extracted into the FI network too.
- Explore objects in the pathway and network views: Object selection in three views has been synchronized. Objects that can be selected include: events in the pathway tree view, objects in the pathway view at the bottom-left corner, and genes and FIs in the network view. You can select an object in one of three views, and corresponding objects in other two views should be selected too. Also you should use features implemented in popup menus in each individual view to explore objects as in a single view.
Note: Using Cytoscape's built-in "Saving Session" feature can save the converted FI networks from pathways. However, displayed pathways cannot be saved into a session file for the time being. We will implement this function in a future release.
Pathway Enrichment Analysis
- Pathway enrichment analysis: A list of genes can be used to check if Reactome pathways have been enriched. To do this, use the popup menu item, "Analyze Pathway Enrichment" (below left figure), to get the dialog for choosing a gene set file (below right figure). You can use a gene set file in one of three file formats: one gene per line, all genes in the same line and delimited by commas, or all genes in the same line and delimited by tabs.
Note: Dependent on the size of your gene list, it may take over 1 minute for running the pathway enrichment analysis. Pathways used in this feature are different from Reactome pathways for annotating a FI network or network modules. Here all about 1,000 pathways have been used. For annotation, only a subset of Reactome pathways, which have been pre-selected for a certain size, are used.
- View enrichment analysis results: Pathway enrichment results are displayed as a table in the "Table Panel" at the bottom of the main Cytoscape window as pathway annotation results. You can use "View in Diagram" to view hit pathways in the pathway diagram view, and use "Export Annotations" to save the results in the table. Pathways in the Reactome pathway tree are highlighted in different colors based on their FDR values. Objects containing genes from your gene list are highlighted in a purple background with a white font in the pathway diagram view. Hit genes are displayed in a thick purple border in the FI network view for a hit pathway.
Note: Hit genes are displayed with same colors in the "Gene List" dialog from the "List Genes" feature.
Probabilistic Graphical Model based Pathway Analysis
Warning: This is an experimental feature. Please use results with caution. The implementation of this feature may change in the future.
We adapted the PARADIGM approach for Reactome pathways by converting reactions drawn in pathway diagrams into factors in factor graphs, a type of probabilistic graphical models (PGMs). For details about the PARADIGM approach, see: Inference of patient-specific pathway activities from multi-dimensional cancer genomics data using PARADIGM. For introduction to factor graphs, see this wikipedia entry: Factor Graph. For test purposes, you can download two sample data files for 100 TCGA ovarian cancer patients: CNVs and mRNA gene expression. The original TCGA OV files were downloaded from the Broad Institute Firehose web site.
- Convert a pathway to a factor graph: As before, open the diagram for a selected pathway. In the pathway diagram panel, use popup menu, "Convert to Graphical Model" to convert a pathway diagram into a factor graph. You can provide a list of small molecules specified by their names that should not be included in the converted factor graph to control the final size of the factor graph by using the following "Escape Names" dialog:
The implementation is based on the PARADIGM approach. A protein in the pathway diagram has been extended into four related nodes in the factor graph based on the central dogma: node for protein activity, node for protein expression, node for protein's mRNA, and node for protein's DNA. The following screenshot shows protein "YAP1" in pathway, YAP1- and WWTR1 (TAZ)-stimulated gene expression, has been converted into four nodes. We use YAP1's DB_ID as labels for its related nodes in the converted factor graph to get a compact display of the factor graph.
You may view pre-assigned values for a selected factor node, which is rendered as a small open rectangle without label in the factor graph view, by using a popup menu, Reactome FI/View Factor Values. The dialog will be popped up to show values for the selected factor (see the dialog at the bottom-right corner in the following screenshot).
- Load observation data: In order to calculate pathway activities for a data set, we need to load observation data. Use popup menu in the factor graph view, Reactome FI/Load Observation Data, to enter a CNV file and a gene expression file using the browse buttons in the "Load Observation Data" dialog (see below). (Note: you may load either a CNV file or a gene expression file, or both). The factor graph inference implemented in this app uses discrete values (three states 0, 1 and 2 for down, normal and up) by discretizing CNV and gene expression values based on the entered threshold values. (Note: you may try our example data files for testing by following links at the top of this section.)
After the observation data is loaded into the factor graph, extra variable nodes (i.e. nodes ending with _mRNA_obs and _DNA_obs) are created and attached to central dogma variable nodes. For example, for YAP1, two observation nodes and two factor nodes linking these observation nodes to central dogma nodes have been added: 1253322_mRNA_obs and 1253322_DNA_obs. The values for these two factors (see two dialogs at the bottom of the following screenshot) have been pre-trained by using the TCGA data sets with the EM algorithm. We will provide features for users to enter factor values and learn these parameters for their own data sets in the future.
Note: You may try some auto-layout feature provided by Cytoscape to make the factor graph viewable after loading observation data.
- Run inference: Use popup menu, Reactome FI/Run Inference, to run inference. You can choose an inference algorithm from the "Inference Algorithm Configuration" dialog and then specify properties for the selected algorithm. We use an open source factor graph C++ library, libdai, for inference. For a detailed explanation about properties for each inference algorithm, please click the "Help" button in the dialog (see below):
It may take a while for performing inference depending on the structure of the factor graph and the sizes of your data files. You may abort the inference if it takes too long. After the inference is finished, you can investigate the results in three places: the IPA Pathway Analysis tab, the IPA Node Values tab, and the Output Analysis Results dialog (the dialog at the top-left corner of the second screenshot below). The two tabs are displayed at the Cytoscape's Table Panel, and the dialog is invoked by clicking the "View Details" button at the top of the IPA pathway analysis tab.
Note: We use the Mann-Whitney U test to calculate p-values for IPA differences between the user's data set and a random data set generated from the user's file in the Output Analysis Results dialog. All other p-values are calculated based on results from 1000 random samples generated from the user's uploaded data files. FDRs are calculated using the Benjamini-Hochberg procedure. Use the popup menu, "Show/Hide Columns for pValues/FDRs" to show or hide p-Values and FDRs (see the above screenshots).
Use the Reactome Functional Interaction (FI) NetworkAfter the Reactome FI Plugin installed, you should see a menu item called "Reactome FI" under the Apps menu. Clicking this menu, you will see 5 sub-menus: Gene Set/Mutation Analysis, HotNet Mutation Analysis, Microarray Data Analysis, Reactome Pathways and User Guide. Gene set/mutation analysis is for doing FI network-based data analysis for a set of genes or a mutation data file, HotNet mutation analysis for the HotNet algorithm to search for network modules (see http://compbio.cs.brown.edu/projects/hotnet/), microarray data analysis for doing MCL (Markov Graph Clustering, http://micans.org/mcl/) based FI network clustering analysis by converting a non-weighted FI network to weighted network using correlations among genes in the network, Reactome pathways for loading pathways from the Reactome database, visualizing Reactome pathways directly in Cytoscape in a their native way, and doing pathway enrichment analysis, and user guide brings you to this user guide.
Gene Set/Mutation Analysis
- Currently FI plug-in supports three file formats for gene set/mutation analysis:
- Simple gene set: one line per gene. For example, GWASFuzzyGenes.txt, a list of T2D GWAS genes.
- Gene/sample number pair. For example, GeneSampleNumber.txt, which contains two required columns, gene and number of samples having gene mutated, and an optional third column listing sample names (delimited by ";").
- NCI MAF (mutation annotation file). For example, GlioblastomaMutationTable.txt, the mutation file from the TCGA GBM project.
- Choose a FI network version from listed three versions.
Note: you may get different results using different FI network versions because a later version may contain more proteins/genes and more FIs. But based on our experience, a significant FI network module is usually stable across multiple versions.
- Choose a file containing genes you want to use to construct a functional interaction network. Select an appropriate file format and parameters to load genes and construct FI network in the dialog. Click the "OK" button to start the FI network building process.
- The constructed FI network will be displayed in the network view panel. A FI specific visual style will be created automatically for the FI network.
- The main features of Reactome FI plug-in should be invoked from a popup menu, which can be displayed by right clicking an empty space in the network view panel.
- Fetch FI annotations: query detailed information on selected FIs. Three FI related edge attribues will be created: FI Annotation, FI Direction, and FI Score. Edges will be displayed based on FI direction attribute values. In the following screenshot, "->" for activating/catalyzing, "-|" for inhibition, "-" for FIs extracted from complexes or inputs, and "---" for predicted FIs. See the "VizMapper" tab, Edge Source Arrow Shape and Edge Target Arrow Shape values for details.
- Analyze network functions: pathway or GO term ennrichment analysis for the displayed network. You can choose to filter enrichment results by a FDR cutoff value. Also you can choose to display nodes in the network panel for a selected row or rows by checking "Hide nodes in not selected rows". The letter in parentheses after each pathway gene set name corresponds to the source of the pathway annotations: C - CellMap, R – Reactome, K – KEGG, N – NCI PID, and B – BioCarta. The following screenshot shows results from a pathway enrichment analysis.
Tip: To analyze pathway or GO term enrichment on a set of genes that are not linked together, select the "Show genes not linked to others" option in the "Set Parameters for FI Network" dialog.
- Cluster FI network: run a network clustering algorithm (spectral partition based network clustering by Newman 2006) on the displayed FI network. Nodes in different network modules will be shown in different colors (different colors used only for first 15 modules based on sizes).
- Analyze module functions: pathway or GO term enrichment analysis for each individual network modules. You can select a size cutoff to filter out network modules that are too small, choose a FDR cutoff to view enriched pathways or GO terms under a certain FDR value, and view nodes in a selected row or rows only in the network diagram.
- Load Cancer Gene Index: load cancer gene index annotations. For details, see section Load Cancer Gene Index.
HotNet Mutation Analysis
Reactome FI Cytoscape plug-in implements the algorithm developed by Raphael's group at Brown University, called "HotNet", for doing cancer mutation data analysis. For details about this algorithm, please see Algorithms for detecting significantly mutated pathways in cancer, and Discovery of mutated subnetworks associated with clinical data in cancer.
- Select a mutation data file and run HotNet algorithm: After selecting sub-menu "HotNet Mutation Analysis" from menu Plugins/Reactome FIs, you would see the following dialog. Choose a version of FI Network, a mutation file from your local file system, and set parameters required by the HotNet algorithm. Currently the plug-in supports the NCI MAF mutation file only. We are going to support more file formats in the future. If you are not sure what delta value should be used, you may choose "Auto" in the dialog. However, using "Auto" takes much longer time to run the algorithm. Random permutation is used to calculate p-values and FDR values. The largest number of permutation is 1000. For details about permutation, please see the above two papers. After entering all required parameters, click the "OK" button to start HotNet analysis. It may take several minutes. If you choose "Auto" for delta, it takes even longer time. For a test run, you may use the TCGA GBM mutation file, GlioblastomaMutationTable.txt, and choose the 2012 version of FI Network with delta 1.0e-4.
- Select network modules and build a FI sub-network: The generated FI network modules from the HotNet analysis are listed in the HotNet result dialog (see below). In the dialog, you can choose a size cutoff, or a FDR cutoff. The displayed selected network modules will be used to build a FI sub-network after you click the "OK" button. In the dialog, you can also see the chosen delta value and the number of permutations. You may try different delta values for better results.
Microarray Data Analysis
The Reactome FI Cytoscape plugin can load gene expression data file, calculate correlations among genes involved in the same FIs, use the calculated correlations as weights for edges (i.e. FIs) in the whole FI network, apply MCL graph clustering algorithm to the weighted FI network, and generate a sub-network for a list of selected network modules based on module size and average correlation. The generated FI sub-network will be displayed in the network panel, and can be used for analysis as in Gene Set/Mutation Analysis. For details about this method, please see our publication: A network module-based method for identifying cancer prognostic signatures.
An array data file should be a tab-delimited text file with table headers. The first column should be gene names. All other columns should be expression values in different samples. The data set in the file should be pre-normalized. For example, see this gene expression file for breast cancer: NejmLogRatioNormGlobalZScore_070111.txt.zip. This data set was download from van de Vijver et al in 2002, and has been normalized.
- Select a microarray data file and run MCL network clustering: After selecting sub-menu "Microarray Data Analysis" from menu Plugins/Reactome FIs, you should see the following dialog. Choose a microarray data file, check if you want to use absolute values as weights for edges, and input an inflation parameter (-I) for the MCL clustering algorithm. The smaller the inflation parameter is, the bigger the average size of generated network modules. Based on our own experience, we use 5.0 for the inflation parameter, the highest recommended value, and choose the absolute value for edge weights. For more details on how to choose the inflation parameter, please see http://micans.org/mcl/. After you have set these parameters, click the OK button to load the data file, calculate correlations, and apply the MCL clustering algorithm.
- Select network modules and build a FI sub-network: The generated network modules are listed in the MCL clustering results dialog (see below). Only modules having more than 2 genes can be listed, and used in the FI sub-network building. You can choose a module size or an average correlation value (absolute value if absolute has been checked before) to filter out modules that may not be significant (Note: after set these cutoff values, please press the "Enter" key to commit your changes.). In our analysis, we choose modules having 7 or more genes with average correlation values no less than 0.25. These values have been used as default in the dialog. In the dialog, you can see how many modules and genes will be chosen for building FI sub-network under your selected filter values. Click the OK button to start the sub-network building. The built sub-network will be displayed, and can be analyzed as with sub-networks generated from the gene set/mutation analysis.
Other Features Related to the FI Network
Query FI SourceSelect an edge and right click it to get the popup menu for edge. Select a menu called "Reactome FI/Query FI Source". If a FI is extracted from curated pathways or reactions, a dialog for the original data source(s) will be displayed. Double click a row in the displayed table to show a detailed web page for the source of the FI. If the selected FI is a predicted one, the evidence for this FI should be displayed.
Fetch FIs for NodeAll FIs for a node can be queried. Select a node in the network panel, and right click it to get the popup menu for node. Select a menu called "Reactome FI/Fetch FIs". FI partners for the selected node will be displayed in two sections: partners have been displayed in the network and partners not displayed in the network. You can select partners from the second sections to expand the displayed network.
Show Pathway DiagramPathway diagrams can be shown for pathway hits. Select a pathway in the "Pathways in Network" or "Pathways in Modules" tab, and right click to get the popup menu for pathway. Select "Show Pathway Diagram" from the popup menu
Load Cancer Gene Index Annotations
Reactome FI plug-in can load NCI cancer gene index annotations for genes/proteins displayed in the network. There are two ways to show these annotations: use a popup menu called "Load Cancer Gene Index" when no object is selected (left figure), and use another popup menu "Fetch Cancer Gene Index" for a selected node (right figure).
By using the first method, the user can load the tree of NCI disease terms and display the tree in the left panel. The user can select disease term in the tree, all genes or proteins have been annotated for the selected disease and its sub-terms will be selected.
By using the second method, the user can view detailed annotations for the selected gene or protein. The user can sort these annotations based on PubMedID, Cancer type, and annotation status, and also filter annotations based on several criteria.
Survival AnalysisSurvival analysis is based on a server-side R script to do either coxph or Kaplan-Meier survival analysis. To do survival analysis, a tab-delimited text file containing at least three columns should be provided. The names of three columns should be: Samples, OSDURATION, and OSEVENT. For example, see this survival information file downloaded from van de Vijver et al in 2002: Nejm_Clin_Simple.txt, which has been simplified for our analysis purpose. To do survival analysis, use the popup menu "Analyze Module Functions/Survival Analysis..." (see below)
In the survival analysis dialog (below), double click the text field to select a file containing survival information for samples used to build the displayed FI sub-network (Note: you cannot do survival analysis if you use a gene set file only to construct the displayed FI subnetweork). You can choose either coxph or Kaplan-Meier model to do survival analysis. If you choose the Kaplan-Meier model, you have to select a module for analysis. In the Kaplan-Meier analysis, all samples will be divided into two groups: samples having no mutated genes in the selected module (group 1) and samples having mutated genes in module (group 2). It is recommended to run the coxph module first without selecting any module in order to see which module is most significantly related to survival times. After that, you can focus on some specific modules for survival analysis.
The results from survival analysis will be displayed in the right Results Panel with a tab labeled "Survival Analysis" (below left). You can do multiple survival analyses. All results returned from the server-side R script will be displayed in this panel with labels based on your parameter selections in the survival analysis dialog. The last result will be selected as default. At most three sections are displayed in the result panel for each analysis: Output, Error, and Plot. If no warning or error returned from an analysis, the error section may not be shown. Rows for modules having p-values less than 0.05 from coxph (all modules) analysis are displayed in blue with text underlined. You can click these modules to do a quick single-module based survival analysis without going through the above steps. Single module-based Kaplan-Meier analysis will show a plot file. You can click the file to view the actual plot (below right). You may need to save the plot file for your future use.
Exporting Data From Reactome
Reactome content can be exported in a number of different formats. For your convenience, export files for the entirety of Reactome are available in the download section, see Download Reactome for details. The details panel for pathway diagrams also provides a way to do exports for individual pathways or even reactions, take a look at Pathway Details. The following export formats are available:
- SBML. An exchange format used by systems biologists for their models. See SBML At Reactome for comprehensive information about SBML export.
- BioPAX. An exchange format used by systems biologists for their models.
- PDF. Text dump of the pathway, organized to look like a research report.
- Word. Text dump of the pathway, organized to look like a research report. The file generated is of type RTF, and can also be understood by OpenOffice and LibreOffice. *Note: RTF files may be very large, but conversion to Word (or PDF) generates a file that is much smaller with no loss of information.
- Protege. A format used for ontology exchange.
- PSI-MOD. A tabular format containing protein-protein interactions derived from Reactome's reactions and complexes. Take a look at PSI-MITAB Interactions for details.
- MySQL. A database dump of all of Reactome's data. To understand the schema properly, please take look at objectrelational-mapping.