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

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.

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

R G Plot for ApoAI Slide 1

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

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)

M A Plot for ApoAI Slide 1

Normalized M A Plot for ApoAI Slide 1

M and A Have Nicer Distributions

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

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.

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).

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 :

limma and limmaGUI Documentation is available after installing the package, by typing “help.start()” in R, clicking on “packages” and then clicking on “limma”. Documentation is available online. Example data sets are available online.

Swirl Zebrafish Example Swirl/ Swirl/