Question: design matrix and lmFit problems in limma
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gravatar for pingzhao Hu
15.5 years ago by
pingzhao Hu210
pingzhao Hu210 wrote:
Hi, I have an Affymetrix dataset. There are 24 arrays. There are three factors: time, sex (M and F) and strain (NOD,R and D4). Sex has two levels and strain has 3 levels. I am only interested in interaction between sex and strain. So the formula will like: ~time+sex*strain. The question I want to ask is " Does the effect of strain change between male and female?" For example I want to select genes: (1) differentially expressed genes between male and female only at NOD not R and D4? (2) differentially expressed genes between male and female only at R not NOD and D4? (3) differentially expressed genes between male and female only at D4 not NOD and R? I construct design and contrast matrix as follow. > design maleNOD maleR maleD4 femaleNOD femaleR femaleD4 [1,] 1 0 0 0 0 0 [2,] 1 0 0 0 0 0 [3,] 0 1 0 0 0 0 [4,] 0 1 0 0 0 0 [5,] 0 0 1 0 0 0 [6,] 0 0 1 0 0 0 [7,] 0 0 0 1 0 0 [8,] 0 0 0 1 0 0 [9,] 0 0 0 0 1 0 [10,] 0 0 0 0 1 0 [11,] 0 0 0 0 0 1 [12,] 0 0 0 0 0 1 [13,] 1 0 0 0 0 0 [14,] 1 0 0 0 0 0 [15,] 0 1 0 0 0 0 [16,] 0 1 0 0 0 0 [17,] 0 0 1 0 0 0 [18,] 0 0 1 0 0 0 [19,] 0 0 0 1 0 0 [20,] 0 0 0 1 0 0 [21,] 0 0 0 0 1 0 [22,] 0 0 0 0 1 0 [23,] 0 0 0 0 0 1 [24,] 0 0 0 0 0 1 > contrast maleNOD-femaleNOD maleR-femaleR maleD4-femaleD4 maleNOD 1 0 0 maleR 0 1 0 maleD4 0 0 1 femaleNOD -1 0 0 femaleR 0 -1 0 femaleD4 0 0 -1 Are these correct???? ----------------------------------------------------------- in lmFit there are three methods to fit linear models (gls,ls and robust). Since affy. data has no duplicate spots for each gene on the same array, Is it still suitable to use gls method???. The example in user manual at 7.2 used gls (default). The big question is that the results based on gls and robust methods are very different: For example, the number of differentially expressed genes based on these two mthods are aso follows (p-value<=0.01): maleNOD-femaleNOD maleR-femaleR maleD4-femaleD4 gls 7 7 14 robust 14 229 500 How can I decide which method (gls or robust) to use? Thanks pingzhao
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