1. A Database of human biological pathways
Steve Jupe -
Please acknowledge Reactome and the sources of Reactome funding (in the panel bottom right).

2. Rationale – Journal information
Nature 407(6805):770-6.The Biochemistry of Apoptosis.
“Caspase-8 is the key initiator caspase in the death-receptor pathway. Upon ligand binding, death receptors such as CD95 (Apo-1/Fas) aggregate and form membrane- bound signalling complexes (Box 3). These complexes then recruit, through adapter proteins, several molecules of procaspase-8, resulting in a high local concentration of zymogen. The induced proximity model posits that under these crowded conditions, the low intrinsic protease activity of procaspase-8 (ref. 20) is sufficient to allow the various proenzyme molecules to mutually cleave and activate each other (Box 2). A similar mechanism of action has been proposed to mediate the activation of several other caspases, including caspase-2 and the nematode caspase CED-3 (ref. 21).”
How can I access the pathway described here and reuse it?
Papers contain rich and useful information, but it is not easily accessible to computers. Languages are highly flexible and nuanced, scientific literature is full of statements that appear definitive but on examination may prove to be based on weak or no cited evidence, or carefully constructed to avoid being too definitive, (a phenomenon known as ‘hedging’). Text mining might spot the red words as ‘molecules’ and blue words as ‘actions’, it might even spot the green possible name of a pathway, and brown literature reference, but to date even the best text mining methods would have trouble identifying the correct relationships.
Reactome data consists of reactions and pathways based on information extracted from papers by people, biologists who are experts in the field, assisted by PhD level curators who add structure to the information and place it in the database. This process maximises the details and subtleties but makes it accessible to data mining and re-use

3. A picture paints a thousand words…
Rationale - Figures
A picture paints a thousand words…
but….
Just pixels
Omits key details
Assumes
Fact or Hypothesis?
A picture is a great way to visualize a process, but impossible for a computer to understand. In most cases you can’t click to obtain more detail. Reactome pathway diagrams are interactive.
Another problem with figures in papers is they assume the reader is familiar with the subject, leaving out some details. Often they mix the established with the hypothetical – Reactome gives the full process in detail, with literature references that experimentally demonstrate the event described.
Nature Oct 12;407(6805):770-6.
The biochemistry of apoptosis.

4. Reactome is… Free, online, open-source curated
database of pathways and reactions in human biology
Authored by expert biologists, maintained by Reactome editorial staff (curators)
Mapped to cellular compartment
Free, unlike most other similar resources, with no restrictions on use (only an acknowledgement is required).
Authored by biologists – NOT simply text-mining (which is prone to misinformation).
Mapped to compartment – not the case for many other pathway dbs.
Tissue and development – too little information to justify when Reactome started, now a recurrent discussion, but not felt to be the primary aim of Reactome – can be achieved by expression data overlay - if no expression, that reaction is likely absent in the tissue or disease.

5. Reactome is… Extensively cross-referenced
Tools for data analysis – Pathway Analysis, Expression Overlay, Species Comparison
Used to infer orthologous events in 17 other species
We link out to many external dbs. Uniprot is our primary external reference for proteins, ChEBI for small molecules, PubMed for literature.
Reactome is human-centric, pathways are based on data obtained using human materials or manually inferred from model organism data, i.e. the expert has to believe that the experiment could be repeated in human with the same results (elaborated in the next slide). In a separate process, human pathways are used to computationally infer (by an orthology method) non-human equivalents.

6. Theory - Reactions = events in biology
Pathway steps = the “units” of Reactome
= events in biology
BINDING
DISSOCIATION
DEGRADATION
CLASSIC
BIOCHEMICAL
PHOSPHORYLATION
DEPHOSPHORYLATION
TRANSPORT
We call steps in a pathway ‘reactions’ an analogy with biochemical pathways. These reactions are the units of Reactome. However, Reactome considers many biological events to be reactions, not just the classical biochemical reaction. Examples are in the slide. Any event that changes the state of a molecule is a reaction.

7. Reaction example 1: Enzymatic
This slide shows a "metabolic" reaction, of the kind that most students will be familiar with. It has reactants (called "inputs" in Reactome- speak), products (outputs) and possibly also a catalyst.
In this real example, ADP is regulating the reaction. More explanation of the symbols used later.

8. Reaction example 2: Transport
Transport of Ca++ from platelet dense tubular system to cytoplasm
R-HSA
This slide illustrates a transport reaction - glucose is transported from the extracellular space to cytosol. The input of this reaction is glucose in the extracellular compartment; the output is glucose in the cytosol. The facilitator is the transporter protein or complex. N.B. In Reactome a molecule in the cytosol is NOT the same as the same molecule in another part of the cell. This is a very important distinction if you want to model events in biology. Reactome uses the GO compartment classification.

9. Other reaction examples
Dimerization
Binding
Differnt kinds of reaction. IRF3 dimerization, complex formation (LBP/LPS/CD14)
N.B. Phosphorylation within Reactome is a reaction, phosphorylated IRF3 is NOT the same as IRF3.
Phosphorylation

10. Reactions connect into Pathways
OUTPUT
INPUT
CATALYST
OUTPUT
INPUT
CATALYST
INPUT
OUTPUT
CATALYST
Pathways are a set of reactions, e.g. Reaction 1: protein X is cleaved, Reaction 2: protein X is phosphorylated, Reaction 3 protein X has catalytic
activity.
Pathways can have sub-pathways to structure information.

11. Evidence – Inferred Reactions
Direct evidence
PMID:5555
PMID:4444
Human pathway
Indirect evidence
PMID:8976
Preference is for direct evidence from human experiments. However, often the evidence is from another species.
In these cases we manually infer the event can happen in humans based on evidence from the other species.
A parallel reaction is created for the non-human and linked to the human reaction.
– this is clearly shown in the db as an ‘Inferred from’ flag in the details of the human reaction.
Nothing to do with the computationally inferred species comparisons!
mouse
PMID:1234
cow

12. Primary external sources
Gene Ontology
Molecular Function
Compartment
Biological Process
ChEBI – small molecules
UniProt – proteins
Ensembl – genes and transcripts
PubMed – literature evidence for events
Molecular Function is used for catalysis
Biological Process is assigned to pathways

13. Curation Version 59 has annotations for: 10212 human proteins,
10214 complexes
~2000 small molecules,
10168 reactions,
2069 pathways,
~25,000 literature references.
With the 52nd quarterly release of Reactome in March 2015, Reactome contains 7,951 human proteins organized into 1,762 pathways, supported by literature references.
If we take a rough figure of 20,000 proteins in the human genome, it might seem that our coverage is low, but bear in mind that between a third and half of those 20,000 have no known function, it is impossible to place them in a pathway if they have no function. When this is taken into account our coverage is approximately half to two-thirds of human proteins with known functions, and growing steadily. If you discover that your favourite pathway or protein is not in Reactome, and want to work with us to see it added, we would be very happy to hear from you!

14. Data Curation Process
Curator
Expert
Reviewer
Pathways authored and reviewed by expert biologists
Curator works with Author to represent knowledge in Reactome data structure
New pathways sent for Review by another expert
New data release every 3 months
Regular Pathway updates.
ORCID and used as attributions for Reactome content
For visibility of expert contributions (authors, reviewers and curators).
Reactome content curation is a slow process, it relies heavily on scientists who generously provide their time and expertise for free, but the result is worth the effort, a gold-standard pathway knowledgebase.
We can’t pay our authors or reviewers, but we do acknowledge their efforts, for significant contributions we can provide Digital Object Identifiers and by using ORCID IDs we register an individual’s contributions to Reactome.

15. Data Expansion – Projecting to Other Species
Human B A
+ ATP A -P
+ ADP
Mouse B A
+ ATP A -P
+ ADP
Drosophila
The basic principle of inference – it’s a little more complicated than this. Details on the website, based on EnsEMBL Compara database.
All OK for mouse, but in fly, protein A has no fly orthologue (using Compara) so the reaction cannot be inferred. Because orthology is harder to establish as the evolutionary distance increases, this process works better with species closer to human like mouse and rat than it does for fly (or yeast). B
Reaction not
projected A
+ ATP
No orthologue - Protein not projected

16. Species Selection
Reactome has computationally predicted pathways across all these species, currently 20.

17. Disease annotation in Reactome
Three main areas:
Infection (eg. HIV, influenza, botulism)
microbially-expressed proteins
Cancer (eg. EGFR, FGFR and NOTCH signalling)
altered protein functions
Metabolic diseases (eg. mucopolysaccharidoses, phenylketonuria, vitamin metabolism abnormalities)
altered expression of proteins
Reactome mostly represents pathways in normal, healthy individuals but is now expanding to include pathways that are associated with disease. There are three main subtypes; infection, cancer and metabolic disorder. Infection represents the interaction between human and microbially-encoded molecules. Cancer is largely the consequence of mutated proteins that have altered function. Metabolic diseases are typically due to mutations that alter expression or the catalytic ability of enzymes.

18. Disease display in Reactome - Infection
Host Interactions of HIV factors
This slide shows how the three subtypes of disease are represented. Top left is an infectious process. Nothing is greyed out, because this process is in one sense a ‘normal’ process, showing the response of the host to the infecting microbe. You can identify that this is a disease process by the use of red boundary lines around the boxes and connecting lines. Top right is an abnormal process associated with cancer. Here the ‘normal’ process is greyed out and abnormal processes that are the consequence of a mutation are highlighted. Bottom left is a metabolic process, where the enzyme is mutated and does not catalyze the reaction, which consequently produces no outputs, indicated by the red crosses on the products of the reaction

19. Disease display in Reactome – Altered protein function in Cancer
Constitutive Signaling by AKT1 E17K in Cancer

20. Disease display in Reactome – Loss of function in metabolism
MPS IIID - Sanfilippo syndrome D

21. Downloads Small(ish) sections from Pathway Browser as text, PDF, etc.
Entire database content (and software) from Downloads page (linked to Homepage).
Reusable standard formats BioPAX SBML
UniProt to Pathways
GO annotations
Protein-Protein interaction pairs - Interactions between proteins in the same complex, reaction, or adjoining reaction

22. Coverage – Content, TOC And many more...
Screenshot of current TOC (accessed via the Menu bar on homepage).
Point is that we have covered lots of topics and pathways, but we always need volunteer authors for topics not covered!

23. Planned Coverage – Editorial Calendar
Release calendar is on the website – it usually gives details of the next 2 releases. Reactome has a quarterly update cycle.

24. Molecular Interaction overlay Species Comparison
Reactome Tools
Interactive Pathway Browser
Analysis
Over-representation
Pathway topology
Expression overlay
Molecular Interaction overlay
Species Comparison
These tools are covered in the tutorial. We often run out of time for Biomart, but it’s in the tutorial.
The pathway browser is the main method of visualizing pathways. Pathway mapping and over-representation are tools to compare a dataset to Reactome content. Expression overlay uses a submitted dataset to colour pathways according to the expression values. Useful to see if the pathway exists in your favourite tissue, or changes expression level in response to some treatment or developmental stage. Molecular interaction overlay adds protein-protein or protein-small molecule interactors onto a pathway. Biomart is a tool for federated queries, i.e. it can join data from more than one database.

25. Tutorial Tutorials available to print out
The tutorials work on the ‘just enough’ principle that just enough detail is given to answer the questions!

26. Front Page http://www.reactome.org
Navigation bar – i.e. A menu bar. Content allows you to explore what is in Reactome, e.g. editorial calendar. Documentation includes the user guide. Tools includes some not in the tutorial, but not all the tools. The most useful ones are now on the Sidebar.
Downloads – e.g. Interactors, the MySQL database.
Sidebar has the most useful tools, and the Search box (top left).
The Search box is explained in the tutorial but not an exercise here. Main point is that it accepts free text, but understands accessions, i.e. If you enter a uniprot ID it will find the protein. Search results return a page of pathways, reactions, proteins and other, mostly literature hits. These are sorted to emphasize pathways and reactions, but across the top of the page are check boxes that allow you to filter by the hit type, e.g. Remove the pathway hits.

27. Search Reactome

28. Search Reactome II

29. Search Reactome III

30. Search Reactome IV

31. Search Reactome V

32. Exercise 1
Answers are in the tutorial document. This exercise is to gain familiarity with the layout of the homepage and investigate some of the menu items.

33. The Pathway Browser Pathway Panel Hierarchy Panel Detail Panel
Analyse Data
Video Tour
Home
Species
Layout
Key
Open
Diagram
Illustrations
Export
Search Diagram
Fit to Page
Pathway
Panel
Hierarchy
Panel
Thumbnail
Zoom/Move
This slide shows the Pathway Browser. This can be reached following a search, or by using the button on the Homepage. There are three main sections. The hierarchy Panel on the left is a hierarchical list of the pathways in Reactome. Many pathways are divided into sub-pathways, which you see as an indentation of the name. Subpathways can be further divided into sub-sub pathways, and again as required, the number of levels depends on how broad the topic is and how many pathways are involved. As you move down the subpathway levels, you will eventually reach levels that show the reactions within the pathway. Selecting the name of any pathway or reaction will cause it to appear or if already visible to be selected, in the Diagram Panel top right, and it will cause details of the event to appear in the Details Panel bottom right.
Note the link to a diagram key top right, the Zoom/Move navigation tools in the top left corner of the Diagram Panel, the Search and Configure icon in the top right of the Diagram Panel, the Thumbnail overview in the bottom left corner of the Diagram Panel. To go back to the Reactome homepage click on the Reactome logo in the top left corner of the page.
Detail Panel

34. Pathway Overview
If you open the Pathway Browser using the Browse Pathways button from the homepage, it displays an overview of all pathways. This Pathway overview is a very useful visualization tool to display the results of an analysis of your experimental data. It is a visualisation of all Reactome pathways in a space-filling graph. The pathways in the hierarchy are represented as rings of subpathway nodes arranged as concentric rings around a central ‘top-level’ node. In effect, the centre of each group is the parent pathway, surrounded by a ring of child subpathways, which are themselves connected to further concentric rings of sub-sub pathways. This overview allows both a quick first glance overview of all pathways, and rapid zooming to interesting results. To open a specific pathway, select and click the node representing the pathway in the overview, or instead go to the pathway hierarchy on the left and select it there.

35. Hierarchy Panel Pathway Reaction Black-box Inferred from New Updated
Disease
The plus/minus buttons reveal/hide more levels. Pathways can have sub-pathways with sub-sub-pathways without limit (though there are plans to limit this), reactions are in pathways, note the little icons.
Black box is sometimes used to avoid breaking an otherwise complete pathway. It has two uses, in the ‘details ommitted deliberately’ sense when the full details would be tedious or impractical to include (e.g. A non-selective DNA ligase) or the reaction is suspected to involve more than is represented and is therefore ‘uncertain’, e.g. Kinase cascades, when an intermediate step has not be eliminated as a possibility. These 2 subtypes have their own symbol in pathway diagrams (explained later).

36. Exercise 2
This exercise is to examine the pathway hierarchy on the left.

37. The Pathway Browser - Pathway Diagrams
Ovals are small molecules (or sets of)
Green boxes are proteins,
Blue are complexes,
Blue with double-boundary are sets
Regulation
+ve ve
Catalyst
Inputs
Outputs
Compartment
Reaction node
Explains (most of) the objects found in a pathway diagram. All objects are interactive – clicking on them will do something, typically show details in the details panel.
Subtypes of reaction with their nodes are based on SBGN (systems biology graphical notation).
Transition Binding Dissociation Omitted Uncertain

38. Exercise 3 …see the Tutorial handouts
This exercise is to look at the objects on the pathway diagram

39. Navigating in the Pathway Browser I
Home button
Click pathway to select, double-click to open diagram
Click reaction to open
Click on a pathway diagram object and it gets the green ring highlight - the equivalent is highlighted in the hierarchy.
These objects with a thick green border are subpathway icons, indicating the existence of a ‘real’ diagram. These are used when the topic is too big for one diagram, or for pathway modules hat can be used in many pathways. To get to the diagram you either click on the highlighted pathway name (recommended as it demonstrates the relationship) or you can right-click and select the option ‘Go To Diagram’
Details here

40. Navigating in the Pathway Browser II
Selected
Highlights
Hover mouse
When looking at a ‘real’ diagram, selecting a part of it (a sub-pathway or reaction) in the hierarchy will highlight the appropriate objects and display the appropriate set of details. Works in reverse too.
Details don’t update until selection

41. Navigating in the Pathway Browser III
When looking at a ‘real’ diagram, selecting a part of it (a sub-pathway or reaction) in the hierarchy will highlight the appropriate objects and display the appropriate set of details. Works in reverse too.

42. Contextual Information Panel
Pin/Unpin
Names/IDs

43. Interactors Click for Interactors Click edge for evidence
Slide to Filter
When looking at a ‘real’ diagram, selecting a part of it (a sub-pathway or reaction) in the hierarchy will highlight the appropriate objects and display the appropriate set of details. Works in reverse too.
Download

44. Exercise 4 …see the Tutorial handouts
Exercise to further establish the connectivity between pathway hierarchy, pathway diagram and details panel.

45. Diagram Search

46. Colour Profiles

47. Diagram Key

48. Export Diagram

49. Show Illustration

50. The Details Panel - Overview
Background
Reveal
Details
Select
Before & After
Orthologues
Explains some of the details in the Detail Panel that are needed for the next exercise.
Key literature

52. Exercise 5 …see the Tutorial handouts
Exercise to look at the Details Panel

53. Disease reaction overlaid on normal pathway
Disease in Reactome
Disease reaction overlaid on normal pathway
Disease details
Click here to display disease reaction

54. Exercise 6 …see the Tutorial handouts
Exercise to look at the Details Panel

55. Understanding gene lists…Reactome Tools
Reactome has several tools that you can use to make sense of large-scale data obtained with high-throughput platform technologies. The next few slides show the tools that will show you how to do this.
The Big List of Differentially Pathways and
Experiment Expressed Molecules beyond…..

56. Analysis

57. Analysis – Identifier list - Data
Pathway analysis button on front page produces this submission form.
Allows import by cut&paste from Excel or browse to a file. Use the Example button for the exercise
DONT FORGET to select the Overrepresentation option – it is not the default!
Click Analyse button to start analysis.

58. Analysis – identifier list - Options
Pathway analysis button on front page produces this submission form.
Allows import by cut&paste from Excel or browse to a file. Use the Example button for the exercise
DONT FORGET to select the Overrepresentation option – it is not the default!
Click Analyse button to start analysis.

59. Analysis Result – Over-representation
FDR
Results – explain the order is ‘best’ at the top

60. Analysis - Pathway topology matching
Results – explain the order is ‘best’ at the top

61. Exercise 7
N.B. Don’t forget the radio button need to be clicked before Analyse (or you get a different result)

62. Expression overlay I
Button on homepage, load example, analyse. It’s an affymetrix microarray dataset.

63. Expression overlay II Scaled to Data Selected
From the results form, click view to see a pathway like this. The colour indicates the expression level, refer to the spectrum bar for values. Right click to zoom into complexes.
The Experiment Browser toolbar steps through multiple columns of expression data (if they were provided).
Step through Data columns

64. Exercise 8

65. Species Comparison I
Button on Homepage Sidebar, form has a dropdown to pick species, then Apply.

66. Species Comparison II Yellow = orthologue No colour = not found
OR no data
Results list all pathways, pick one and it looks a bit like this – colours overlaid onto the pathway diagram. Box on right shows the result of right-clicking a complex to expand it and see the components in a grid.

67. Exercise 9 …see the Tutorial handouts
This exercise is to examine species comparison.

68. Molecular Interaction Overlay
Zoom
Click line…

69. Molecular Interaction Overlay - Data

70. Molecular Interaction Overlay – Set source
MI overlay doesn’t have a button on the homepage, RIGHT-click on a protein object in the pathway diagram and select display interactors
Explain first diagram for Syk – 6 interactors, could show up to 10, numbers go up to 50 then say 50+, can see up to 50 in table (explained later) possible to export more (later).
Explain second diagram – can add interactors to many proteins, interactors can connect to more than one protein rather than drawn twice.
AA&U button produces a Config panel
Click Display table - does as it says, up to 50 per protein on the pathway diagram.
In the table, clicking a blue square adds/removes the interactors from the named protein, clicking a name re-centres the diagram on that object.
Export fetches all interactors from the source db, not just 50.
You can colour interactors by confidence score.
If you change the Interaction database, any displayed interactions are removed and the interactions redrawn according to data from the new source. ChEBI fetches small molecules rather than protein interactors.

71. Exercise 10
…see the Tutorial handouts

72. More Information From the Reactome Homepage
Reactome User Guide
Tour
Train Online -
For more information about Reactome please refer to the Reactome User Guide, or ask us a question via the help link on the homepage, or go to the Train Online system and look at the Reactome courses there.

73. Acknowledgements
Reactome is based at the EBI in the UK, OICR in Toronto, Canada, and New York University School of Medicine, USA.

74. Questions?