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Gene Set Enrichment Analysis Genome 373 Genomic Informatics Elhanan Borenstein

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Page 1: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Gene Set Enrichment Analysis

Genome 373

Genomic Informatics

Elhanan Borenstein

Page 2: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Gene expression profiling

Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response, etc.)

The Gene Ontology (GO) Project

Provides shared vocabulary/annotation

Terms are linked in a complex structure

Enrichment analysis:

Find the “most” differentially expressed genes

Identify over-represented annotations

Modified Fisher's exact test

A quick review

Page 3: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Enrichment Analysis ClassA ClassB

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Cutoff

Biological function?

Page 4: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Enrichment Analysis ClassA ClassB

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Page 5: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

After correcting for multiple hypotheses testing, no individual gene may meet the threshold due to noise.

Alternatively, one may be left with a long list of significant genes without any unifying biological theme.

The cutoff value is often arbitrary!

We are really examining only a handful of genes, totally ignoring much of the data

Problems with cutoff-based analysis

Page 6: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

MIT, Broad Institute

V 2.0 available since Jan 2007

Gene Set Enrichment Analysis

(Subramanian et al. PNAS. 2005.)

Page 7: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Calculates a score for the enrichment of a entire set of genes rather than single genes!

Does not require setting a cutoff!

Identifies the set of relevant genes as part of the analysis!

Provides a more robust statistical framework!

GSEA key features

Page 8: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Gene Set Enrichment Analysis ClassA ClassB

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Page 9: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Gene Set Enrichment Analysis ClassA ClassB

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Running sum: Increase when gene is in set

Decrease otherwise

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Page 10: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Gene Set Enrichment Analysis

What would you expect if the hits were randomly distributed?

What would you expect if most of the hits cluster at the top of the list?

Page 11: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Gene Set Enrichment Analysis

Genes within functional set

(hits)

Running sum

Enrichment score (ES) = max deviation from 0

Leading Edge genes

Page 12: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Gene Set Enrichment Analysis Low ES (evenly distributed) ES = 0.43 ES = -0.45

Page 13: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

Ducray et al. Molecular Cancer 2008 7:41

Gene Set Enrichment Analysis

Page 14: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

1. Calculation of an enrichment score (ES) for each functional category

2. Estimation of significance level of the ES

An empirical permutation test

Phenotype labels are shuffled and the ES for this functional set is recomputed. Repeat 1000 times.

Generating a null distribution

3. Adjustment for multiple hypotheses testing

Necessary if comparing multiple gene sets (i.e.,functions)

Computes FDR (false discovery rate)

GSEA Steps

Page 15: Gene Set Enrichment Analysis - elbo.gs.washington.edu...Gene expression profiling Which molecular processes/functions are involved in a certain phenotype (e.g., disease, stress response,

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