Dear list and Gordon,

I have some troubles to computed a moderated paired t-test in the linear model.
Here is my experimental plan :

I used a single channel Agilent microarray.
2 types of cells : Control (S) and Treated (T)
Fives human donors : 4-5-6-7-8
Two times of treatment : 4 hours and 18 hours

I want to compare teh differential expresed genes between my C versus T at 4 hours and then at 18 hours.

Here is my design :


My targets frame is :
>  Targets
          X                                           FileName Treatment Donor Time
1   DC_4_4 US10463851_252665214446_S01_GE1_1010_Sep10_1_2.txt         T     4    4
2   SC_4_4 US10463851_252665214448_S01_GE1_1010_Sep10_1_2.txt         C     4    4
3  DC_18_4 US10463851_252665214447_S01_GE1_1010_Sep10_1_2.txt         T     4   18
4  SC_18_4 US10463851_252665214444_S01_GE1_1010_Sep10_1_3.txt         C     4   18
5   DC_4_5 US10463851_252665214448_S01_GE1_1010_Sep10_1_4.txt         T     5    4
6   SC_4_5 US10463851_252665214444_S01_GE1_1010_Sep10_1_1.txt         C     5    4
7  DC_18_5 US10463851_252665214446_S01_GE1_1010_Sep10_1_3.txt         T     5   18
8  SC_18_5 US10463851_252665214447_S01_GE1_1010_Sep10_1_4.txt         C     5   18
9   DC_4_6 US10463851_252665214445_S01_GE1_1010_Sep10_1_4.txt         T     6    4
10  SC_4_6 US10463851_252665214447_S01_GE1_1010_Sep10_1_3.txt         C     6    4
11 DC_18_6 US10463851_252665214448_S01_GE1_1010_Sep10_1_3.txt         T     6   18
12 SC_18_6 US10463851_252665214445_S01_GE1_1010_Sep10_1_3.txt         C     6   18
13  DC_4_7 US10463851_252665214444_S01_GE1_1010_Sep10_1_4.txt         T     7    4
14  SC_4_7 US10463851_252665214445_S01_GE1_1010_Sep10_1_2.txt         C     7    4
15 DC_18_7 US10463851_252665214447_S01_GE1_1010_Sep10_1_1.txt         T     7   18
16 SC_18_7 US10463851_252665214446_S01_GE1_1010_Sep10_1_1.txt         C     7   18
17  DC_4_8 US10463851_252665214444_S01_GE1_1010_Sep10_1_2.txt         T     8    4
18  SC_4_8 US10463851_252665214446_S01_GE1_1010_Sep10_1_4.txt         C     8    4
19 DC_18_8 US10463851_252665214445_S01_GE1_1010_Sep10_1_1.txt         T     8   18
20 SC_18_8 US10463851_252665214448_S01_GE1_1010_Sep10_1_1.txt         C     8   18


then I create my design matrix :

>  Donor
  [1] 4 4 4 4 5 5 5 5 6 6 6 6 7 7 7 7 8 8 8 8
Levels: 4 5 6 7 8
>  Treat=factor(Targets$Treatment,levels=c("C","T"))
>  Treat
  [1] T C T C T C T C T C T C T C T C T C T C
Levels: C T
>  Time=Treat=factor(Targets$Time)
>  Time
  [1] 4  4  18 18 4  4  18 18 4  4  18 18 4  4  18 18 4  4  18 18
Levels: 4 18

>  design=model.matrix(~Donor+Treat+Time)
>  design
    (Intercept) Donor5 Donor6 Donor7 Donor8 Treat18 Time18
1            1      0      0      0      0       0      0
2            1      0      0      0      0       0      0
3            1      0      0      0      0       1      1
4            1      0      0      0      0       1      1
5            1      1      0      0      0       0      0
6            1      1      0      0      0       0      0
7            1      1      0      0      0       1      1
8            1      1      0      0      0       1      1
9            1      0      1      0      0       0      0
10           1      0      1      0      0       0      0
11           1      0      1      0      0       1      1
12           1      0      1      0      0       1      1
13           1      0      0      1      0       0      0
14           1      0      0      1      0       0      0
15           1      0      0      1      0       1      1
16           1      0      0      1      0       1      1
17           1      0      0      0      1       0      0
18           1      0      0      0      1       0      0
19           1      0      0      0      1       1      1
20           1      0      0      0      1       1      1
attr(,"assign")
[1] 0 1 1 1 1 2 3
attr(,"contrasts")
attr(,"contrasts")$Donor
[1] "contr.treatment"

attr(,"contrasts")$Treat
[1] "contr.treatment"

attr(,"contrasts")$Time
[1] "contr.treatment"


In this design matrix I think something is wrong, because of the column Treat18 is the same as Time18.
I don't understand why.
So, the following code failed, and the differential expressed genes are odds.

Somebody can help me !!! Thanks all.


>  fit=lmFit(test_norm,design)
Coefficients not estimable: Time18
Message d'avis :
Partial NA coefficients for 34183 probe(s)
>  fit2=eBayes(fit)
Message d'avis :
In ebayes(fit = fit, proportion = proportion, stdev.coef.lim = stdev.coef.lim,  :
   Estimation of var.prior failed - set to default value


>  table = topTable(fit2,1, number=5000, p.value=0.05,adjust.method="BH",sort.by="logFC",lfc=2)
>  head(table)
                  ID    logFC  AveExpr         t      P.Value    adj.P.Val        B
6509  A_33_P3396434 18.44159 18.41239 245.14490 1.308161e-31 2.353520e-28 53.41519
22398 A_33_P3223592 18.25824 18.24591 242.75647 1.545005e-31 2.514901e-28 53.36821
10771 A_33_P3244165 18.21029 18.02229  90.76191 2.796577e-24 2.467615e-23 44.59915
6149  A_33_P3346552 18.14780 18.12098 207.18556 2.282464e-30 1.147374e-27 52.50960
23554 A_33_P3210160 18.08158 18.21026 239.64192 1.924175e-31 2.560908e-28 53.30521
20924 A_33_P3286278 18.04425 18.07312 179.72121 2.558128e-29 5.025546e-27 51.56876


Best,

Ingrid


-- 
Ingrid MERCIER
Mycobacterial Interactions with Host Cells Team
Institute of Pharmacology&  Structural Biology
CNRS - University of Toulouse
BP 64182
F-31077 Toulouse Cedex France
Tel +33 (0)5 61 17 54 63




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