Pathway analysis Daniel Hurley

1. Pathway analysisDaniel Hurley

2. Pathway analysis: summary A popular buzzword… but what does it mean? How do you do it? How do you interpret the results?

3. First

4. What we mean by ‘pathway analysis’ • A ‘pathway’ implies causation, but don’t be fooled!

5. What we mean by ‘pathway analysis’ • A ‘pathway’ implies causation, but don’t be fooled! • Most ‘pathway analysis’ actually identifies groups of functionally similar transcripts. • Louis’ example: (http://gather.genome.duke.edu/)

6. What we mean by ‘pathway analysis’ • A useful paper…. But the conclusion is: lots of tools, some quite different approaches!

7. What we mean by ‘pathway analysis’ • Pathway analysis tools like GATHER, DAVID, and GeneSetDB typically rely on enrichment analyses to tell us things. • This set of techniques asks the question ‘of this set of genes, how many share any particular function, and is that more than we would expect by chance?’ • Example: the top 200 most differentially-expressed genes by some ranking (e.g. adjusted p-value) • Determination of ‘by chance’ is usually done using a permutation (= Monte Carlo) approach • Other ‘pathway analyses’ involve signatures of groups of transcripts (e.g. using Principal Component Analysis)

8. What we mean by ‘pathway analysis’ But what do we mean by a ‘function’? Lots of things: Protein function Hypothetical protein function Chromosomal location Metabolic pathway association Disease association

9. Daunting

10. The key point

11. What we mean by ‘pathway analysis’ • Pathway analysis can identify common features present in a group of transcripts • What the output means depends on the specific biology under study • No such thing really as a ‘general’ pathway analysis • A good place to start is by finding papers relevant to the specific biology

12. What can you do with it? Some answers: • Get a general picture of the active functions in a condition (vs. control) • Identify conditions with similar functions • Investigate whether a particular function is active in a condition • Investigate the functions associated with a particular gene • Differentiate conditions by their active functions

13. Next

14. Pathway analysis: how you do it • Begin with a list of transcripts of interest

15. Pathway analysis: how you do it • Choose a web-based tool: GATHER, DAVID and GeneSetDB are good ones to start But Pathguide.org has 325 pathway links at last count

16. Pathway analysis: how you do it • Enter the list of transcripts: with most tools, you will either paste in gene names or identifiers, or upload a file

17. Finally

18. Pathway analysis: interpreting results • Basic tools will produce ranked lists of the most ‘enriched’ categories: GATHER

19. Pathway analysis: interpreting results • More sophisticated ones will produce ‘network’ diagrams DAVID Ingenuity Pathways Analysis But the interpretation of these is rather subjective

20. Summary • Pathway analysis should probably be called information enrichment analysis – a more accurate term • Used prudently, it is a useful tool for exploring the functional landscape of an experiment • To make it meaningful, you need to interpret the results in the context of the specific biology under study • There are a lot of web-based tools; start with one which is current and produces a result you value • To start, you need a set of (transcripts) of interest • To present the results, you can use a simple table, or a more complex ‘network’ diagram • Risk: false-positives are very difficult to identify, and with enough data you can link any molecular species to any other species

21. Fin Any questions?