Dear Gordon,
   
  I forgot to mention in my previous e-mail that all QCs were fine and MA plots created with the MAplot function were absolutely normal.
  I think it is an essential comment.
  THANK you,
  Lev.

Lev Soinov <lev_embl1@yahoo.co.uk> wrote:
  Dear Gordon,

We are analysing a dataset of 14920 genes obtained with the AB1700 platform.
It has three treatments L1, L2, L1+L2 and control. The data is in the form of expression data matrix with the first column as pobe ID and 14 other columns correspond to 4 above conditions. Using the code below, we obtain a huge number of genes with adjusted p values <0.05, about 5000 for the comparison between L1 and control for example. At the same time B values corresponding to these probes are very small, i.e. we are getting B<-4 in the bottom of the list of probes with adj.p<0.05.
Could you please comment on possible causes for this? Is it normal?
With kind regards,
Lev.

>s<-scan('Data.txt',what='character')
Read 223800 items
> sm<-matrix(s,byrow=TRUE,ncol=15)
> dim(sm)
[1] 14920    15
> rownames(sm)<-sm[,1]
> sm<-sm[,2:ncol(sm)]
> snn<-apply(sm,2,as.numeric)
> rownames(snn)<-rownames(sm)
> signals<-snn
> dim(signals)
[1] 14920    14
> temp<-normalizeBetweenArrays(log2(signals), method='quantile')
> design <- model.matrix(~0 +factor(c(1,1,1,1,2,2,2,3,3,3,3,4,4,4)))
> colnames(design) <- c("Control","L1","L2","L1L2")
> contrast.matrix <- makeContrasts(L1-Control,L2-Control,L1L2-Control,levels=design)
> fit <- lmFit(temp, design)
> fit2 <- contrasts.fit(fit, contrast.matrix)
> fit2 <- eBayes(fit2)
> topTable(fit2, coef=1, adjust='BH')
          ID    logFC        t      P.Value    adj.P.Val        B
8790  182417 5.813459 38.16912 1.072876e-15 1.479057e-11 25.47446
6945  165482 8.573261 35.59768 2.856130e-15 1.479057e-11 24.69238
7132  167208 6.247484 35.49523 2.973975e-15 1.479057e-11 24.65950
10941 202780 4.881978 33.98673 5.467499e-15 2.039377e-11 24.15906
1102  109858 5.076380 33.01348 8.214210e-15 2.451120e-11 23.81910
3785  135458 3.686867 32.09869 1.217373e-14 3.027202e-11 23.48654
12355 215284 5.035617 30.68240 2.288454e-14 4.877676e-11 22.94512
6515  161539 5.885744 29.64292 3.704033e-14 6.908021e-11 22.52582
9789  191745 8.189347 28.65188 5.953817e-14 9.870106e-11 22.10752
8568  180293 4.749725 27.88955 8.671664e-14 1.293812e-10 21.77270


The bottom of the adj.p<0.05 list:
        ID      logFC               t                P.Value         adj.P.Val        B
207673  -0.293302      -2.70223       0.01703      0.042251  -4.2848
213498  -0.675519      -2.70219      0.017033     0.042251  -4.2849
186148  -0.419934      -2.70201      0.017039     0.042258  -4.2853
233859  -0.422533      -2.70185      0.017044     0.042263  -4.2856
188263  -0.330067      -2.70179      0.017046     0.042263  -4.2857

    
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