1. limmaGUI A Point-and-Click Interface for cDNA Microarray Analysis James Wettenhall and Gordon Smyth Division of Genetics and Bioinformatics Walter and Eliza Hall Institute of Medical Research

2. limma, limmaGUI and affylmGUI limma : linear models for microarrays by Gordon Smyth Also contains many useful functions specifically for cDNA microarrays limmaGUI : A Graphical User Interface for cDNA analysis with limma. affylmGUI : A Graphical User Interface for Affymetrix analysis with limma.

4. R, G, M and A R f = Red Foreground Intensity R b = Red Background Intensity R = R f - R b G f = Green Foreground Intensity G b = Green Background Intensity G = G f - G b

5. R G Plot for ApoAI Slide 1

6. log 2 (R) log 2 (G) Plot for ApoAI Slide 1

7. M and A Log Ratio : M (“Minus”) = log 2 (R/G) = log 2 R – log 2 G Average Log Intensity : A (“Add”) = log 2 (RG) 1/2 = (1/2)(log 2 R + log 2 G)

8. M A Plot for ApoAI Slide 1

9. Normalized M A Plot for ApoAI Slide 1

10. M and A Have Nicer Distributions

11. Linear Models in Microarrays Suppose for one gene, we have: RG Array 14 (KO)32 (WT) Array 215 (WT)2 (KO) M 1 = log 2 (R 1 /G 1 ) = log 2 (4/32) = -3 M 2 = log 2 (R 2 /G 2 ) = log 2 (15/2) = 2.9

12. Linear Models in Microarrays This linear model has one parameter, M KO-WT to be estimated for each gene. This parameter was estimated using a simple (weighted) average. A factor of (-1) was used for the dye-swap.

13. Linear Models in Microarrays What about confidence statistics? As M 1 is close to -M 2, we are confident in our estimate for M KO-WT so we expect: A low p-value A high B statistic (log-odds of D.E.) A large negative moderated t statistic (because this gene is down-regulated).

14. Linear Models in Microarrays What makes this a LINEAR model? Let E{ } be the expected value of. We have : E{M 1 } = (1) M KO-WT E{M 2 } = (-1) M KO-WT A linear relationship. The design matrix is :

15. limma and limmaGUI http://bioinf.wehi.edu.au/limma Documentation is available after installing the package, by typing “help.start()” in R, clicking on “packages” and then clicking on “limma”. http://bioinf.wehi.edu.au/limmaGUI Documentation is available online. Example data sets are available online.

16. Swirl Zebrafish Example http://bioinf.wehi.edu.au/limmaGUI/Doc/ Swirl/http://bioinf.wehi.edu.au/limmaGUI/Doc/ Swirl/