Gene Expression Analysis Web Forum. Jonathan Gerstenhaber Field Application Specialist

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1 Gene Expression Analysis Web Forum Jonathan Gerstenhaber Field Application Specialist

2 Our plan today: Import Preliminary Analysis Statistical Analysis Additional Analysis Downstream Analysis 2 Copyright Partek Inc.

3 Custom Data Import Data and annotation should be in two separate files Data should be 1 sample per row, 1 gene per column. Optional annotation file should be 1 gene per row, 1 annotation per column The first column, ProbeID, should match with the data headers The column with Hugo ID s should be named Gene Symbol 3 Copyright Partek Inc.

4 REML versus MoM Strictly speaking MoM is a better model because it will result in unbiased estimates of random and fixed effects Unfortunately, in unbalanced or incomplete data, MoM will often fail to run 4 Copyright Partek Inc.

5 REML versus MoM One way ANOVA Two way MoM ANOVA = On all samples = On paired samples Two way REML ANOVA 5 Copyright Partek Inc.

6 Real data example In this data set some of our subjects only contributed a single tissue For these subjects, the tissue and subject factors are confounded To properly account for subject effects, these samples will have to be ignored Tissue\ Sample Astrocyte Cerebellum Cerebrum Heart Copyright Partek Inc.

7 How ANOVA sees the data Using the batch effect remover, we can how ANOVA sees it when computing tissue significance Original data Where did the 2 extra samples go? They perfectly overlap at the group mean The samples are effectively being ignored Sample corrected 7 Copyright Partek Inc.

8 Biological Interpretation Biological relevance is not usually found in only a single gene Most often biological changes are made through concerted modifications in many genes in parallel. Concerted effect on what? Gene products Functional Categories Metabolic Pathways 8 Copyright Partek Inc.

9 Biological Interpretation 3 Steps 1. Group genes into biologically relevant categories Default is Gene Ontology Custom files can be used as well 2. Test to see if our lead genes act together in one or a few of these categories Gene Set Enrichment 3. Test the categories individually for biologically relevant changes GO ANOVA GSEA 9 Copyright Partek Inc.

10 Custom Gene Sets Build your own following the GMT format One row per gene set First two columns name and define that set Followed by a list of genes Or download one off of the web! The Broad s MSigDB has several available in the GMT format 10 Copyright Partek Inc.

11 GO Enrichment Test lead genes for a common biological denominator Check to see if the genes found to be differentially expressed fall into a category more often than expected by chance. Significant genes in a category All significant genes vs All genes in the category All genes on the chip This is called the enrichment score Calculated from a 2x2 Fisher s exact test We will also get the ability to look at fold changes and average p-values 11 Copyright Partek Inc.

12 Gene Set ANOVA This analysis can be run prior to any gene specific analysis as it uses all genes to get an estimate of function group expression In the case of differential expression, each sample s expression of a gene set is taken from the average of all the genes 12 Copyright Partek Inc.

13 Gene Set ANOVA Sometimes we are not expecting an entire function category to change Example: G1->S checkpoint is compromised, often the G2->M checkpoint is strengthened In this case what we are interested in is disruption the pathway Calculated from the interaction of the gene with a variable Does cancer change the pattern of gene expression within the gene set? AQP1 is the dominant water transporter for muscle AQP4 is specific to neural cells 13 Copyright Partek Inc.

14 GSEA GSEA can use either correlation or a signal to noise ratio to rank genes Can only look at a single variable It then looks at the high ranked genes for an enrichment score Includes FDR and p- value as well 14 Copyright Partek Inc.

15 Upcoming Online Events Webinars (partek.com/webinars) 25 Mar Focus on: Analysis of Next Generation Sequencing Data User Forums (Help->Online Tutorials) 22 Apr Copy Number User Forum 15 Copyright Partek Inc.

16 Summary 6.5 has introduced more streamlined list creation GX analysis can extend past the workflow You can always ask us questions at: 16 Copyright Partek Inc.