[BioC] design matrix Limma design for paired t-test
Belinda Phipson
phipson at wehi.EDU.AU
Wed Jun 13 08:45:45 CEST 2012
Hi Ingrid
The problem with your code is the following line:
> Time=Treat=factor(Targets$Time)
Where you essentially set the time factor equal to the treat factor.
Cheers,
Belinda
-----Original Message-----
From: bioconductor-bounces at r-project.org
[mailto:bioconductor-bounces at r-project.org] On Behalf Of Ingrid Mercier
Sent: Wednesday, 13 June 2012 1:02 AM
To: bioconductor at r-project.org; smyth at wehi.edu.au
Subject: [BioC] design matrix Limma design for paired t-test
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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