Pathway analysis

Daniel Hurley

Pathway analysis: summary

A popular buzzword…but what does it mean?

How do you do it?

How do you interpret the results?

First

What we mean by ‘pathway analysis’

• A ‘pathway’ implies causation, but don’t be fooled!

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/)

• A useful paper….

What we mean by ‘pathway analysis’

But the conclusion is: lots of tools, some quite different

approaches!

• 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)

What we mean by ‘pathway analysis’

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

Daunting

The key point

• 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

What we mean by ‘pathway analysis’

What can you do with it?Some answers:

Get a general picture of the

active functions in a condition (vs.

control)

Investigate whether a particular

function is active in a condition

Differentiate conditions by their active functions

Investigate the functions

associated with a particular gene

Identify conditions with

similar functions

Next

• Begin with a list of transcripts of interest

Pathway analysis: how you do it

• Choose a web-based tool: GATHER, DAVID and GeneSetDB are good ones to start

Pathway analysis: how you do it

But Pathguide.org has 325 pathway links at last count

• Enter the list of transcripts: with most tools, you will either paste in gene names or identifiers, or upload a file

Pathway analysis: how you do it

Finally

• Basic tools will produce ranked lists of the most ‘enriched’ categories:

Pathway analysis: interpreting results

GATHER

• More sophisticated ones will produce ‘network’ diagrams

Pathway analysis: interpreting results

DAVID

Ingenuity Pathways AnalysisBut the interpretation of these

is rather subjective

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

FIN

Any questions?