Entering edit mode
Hi Agnieszka,
Neither approach is more correct than the other, but the second is
very
likely to be more powerful.
In either case the numerator of the contrast will be the same.
However,
the denominator of the contrast is based on the intra-group
variability
for all of the groups in your linear model. So if you only include the
first treatment comparison in your model then the denominator will be
based on just those two groups. If you use all 20 of your slides, then
you increase the amount of data that can be used to compute the
intra-group variability.
Since the accuracy of a variance estimate is dependent on the amount
of
data (it gets more accurate as you increase the amount of data used),
you will probably have more power to detect differences if you include
all the data in your linear model. And more power is usually
considered
to be better.
Best,
Jim
James W. MacDonald, M.S.
Biostatistician
Hildebrandt Lab
8220D MSRB III
1150 W. Medical Center Drive
Ann Arbor MI 48109-0646
734-936-8662
>>> Agnieszka Zmienko <akisiel at="" ibch.poznan.pl=""> 02/17/09 9:05 AM >>>
Hi!
I am "pure" biologist so I strictly follow the
limma userguide commands in my analysis but I have a problem.
I have a set of microarrays with a common control
channel. I have 4 biological replicates of each
experiment. If I perform the simplest possible
analysis for the first treatment
(wt.NaCl/wt.pool) using only files
043,046,048,077 as targets ("targetsA.txt"), I
obtain different adjusted p.values (and different
topTable) comparing to analysis with all twenty
input files ("targetsB.txt") and extracting
wt.NaCl contrast. Why? Which topTable is correct?
Agnieszka
Here are my targets files and the codes:
TargetsA.txt
SlideNumber Name FileName Cy3 Cy5
43 wt.Na1 043.gpr wt.pool wt.NaCl
46 wt.Na2 046.gpr wt.pool wt.NaCl
48 wt.Na3 048.gpr wt.pool wt.NaCl
77 wt.Na4 077.gpr wt.pool wt.NaCl
TargetsB.txt
SlideNumber Name FileName Cy3 Cy5
43 wt.Na1 043.gpr wt.pool wt.NaCl
46 wt.Na2 046.gpr wt.pool wt.NaCl
48 wt.Na3 048.gpr wt.pool wt.NaCl
77 wt.Na4 077.gpr wt.pool wt.NaCl
44 wt.Cd1 044.gpr wt.pool wt.CdCl2
47 wt.Cd2 047.gpr wt.pool wt.CdCl2
49 wt.Cd3 049.gpr wt.pool wt.CdCl2
78 wt.Cd4 078.gpr wt.pool wt.CdCl2
75 mu1.U1 075.gpr wt.pool mu1.U
70 mu1.U2 070.gpr wt.pool mu1.U
71 mu1.U3 071.gpr wt.pool mu1.U
80 mu1.U4 080.gpr wt.pool mu1.U
67 mu2.U1 067.gpr wt.pool mu2.U
74 mu2.U2 074.gpr wt.pool mu2.U
79 mu2.U3 079.gpr wt.pool mu2.U
68 mu2.U4 068.gpr wt.pool mu2.U
72 mu3.U1 072.gpr wt.pool mu3.U
73 mu3.U2 073.gpr wt.pool mu3.U
69 mu3.U3 069.gpr wt.pool mu3.U
88 mu3.U4 088.gpr wt.pool mu3.U
> targetsA=readTargets("targetsA.txt")
>
RGA=read.maimages(targetsA,source="genepix",wt.fun=wtflags(weight=0,
cutoff=-50))
Read 043.gpr
Read 046.gpr
Read 048.gpr
Read 077.gpr
> spottypes=readSpotTypes("SpotTypes.txt")
> RG$genes$Status=controlStatus(spottypes,RGA)
Matching patterns for: ID Name
Found 31200 cDNA
Found 48 no_change
Setting attributes: values Color
> RGAb=backgroundCorrect(RGA, method="normexp", offset=50)
Green channel
Corrected array 1
Corrected array 2
Corrected array 3
Corrected array 4
Red channel
Corrected array 1
Corrected array 2
Corrected array 3
Corrected array 4
> MAA=normalizeWithinArrays(RGAb)
> fitA=lmFit(MAA)
Warning message:
In lmFit(MAA) :
Some coefficients not estimable: coefficient interpretation may
vary.
> fitA=eBayes(fitA)
> write.table(topTable(fitA,
+
number=100,adjust.method="BH",p.value=0.05,lfc=1,sort.by="P",resort.by
="logFC"),"topTableA.txt")
> write.fit(fitA, digits=6,F.adjust="BH",file="resultsA.txt")
---------------------------------
> targetsB=readTargets("targetsB.txt")
>
RGB=read.maimages(targetsB,source="genepix",wt.fun=wtflags(weight=0,
cutoff=-50))
Read 043.gpr
Read 046.gpr
Read 048.gpr
ReRead 074.gpr
Read 079.gpr
Read 068.gpr
Read 072.gpr
Read 073.gpr
Read 069.gpr
Read 088.gpr
> spottypes=readSpotTypes("SpotTypes.txt")
> RG$genes$Status=controlStatus(spottypes,RGB)
Matching patterns for: ID Name
Found 31200 cDNA
Found 48 no_change
Setting attributes: values Color
> RGBb=backgroundCorrect(RGB, method="normexp", offset=50)
Green channel
Corrected array 1
Corrected array 2
Corrected array 3
Corrected array 4
Corrected array 5
Corrected array 6
Corrected array 7
Corrected array 8
Corrected array 9
Corrected array 10
Corrected array 11
Corrected array 12
Corrected array 13
Corrected array 14
Corrected array 15
Corrected array 16
Corrected array 17
Corrected array 18
Corrected array 19
Corrected array 20
Red channel
Corrected array 1
Corrected array 2
Corrected array 3
Corrected array 4
Corrected array 5
Corrected array 6
Corrected array 7
Corrected array 8
Corrected array 9
Corrected array 10
Corrected array 11
Corrected array 12
Corrected array 13
Corrected array 14
Corrected array 15
Corrected array 16
Corrected array 17
Corrected array 18
Corrected array 19
Corrected array 20
> MAB=normalizeWithinArrays(RGBb)
> designB=modelMatrix(targetsB,ref="wt.pool")
Found unique target names:
mu1.U mu2.U mu3.U wt.CdCl2 wt.NaCl wt.pool
> designB
mu1.U mu2.U mu3.U wt.CdCl2 wt.NaCl
[1,] 0 0 0 0 1
[2,] 0 0 0 0 1
[3,] 0 0 0 0 1
[4,] 0 0 0 0 1
[5,] 0 0 0 1 0
[6,] 0 0 0 1 0
[7,] 0 0 0 1 0
[8,] 0 0 0 1 0
[9,] 1 0 0 0 0
[10,] 1 0 0 0 0
[11,] 1 0 0 0 0
[12,] 1 0 0 0 0
[13,] 0 1 0 0 0
[14,] 0 1 0 0 0
[15,] 0 1 0 0 0
[16,] 0 1 0 0 0
[17,] 0 0 1 0 0
[18,] 0 0 1 0 0
[19,] 0 0 1 0 0
[20,] 0 0 1 0 0
> fitB=lmFit(MAB,designB)
Warning message:
In lmFit(MAB, designB) :
Some coefficients not estimable: coefficient interpretation may
vary.
> contrast.matrix=makeContrasts(wt.NaCl,levels=designB)
> contrast.matrix
Contrasts
Levels wt.NaCl
mu1.U 0
mu2.U 0
mu3.U 0
wt.CdCl2 0
wt.NaCl 1
> fitB=contrasts.fit(fitB,contrast.matrix)
> fitB=eBayes(fitB)
> write.table(topTable(fitB,
+
number=100,adjust.method="BH",p.value=0.05,lfc=1,sort.by="P",resort.by
="logFC"),"topTableB.txt")
> write.fit(fitB, digits=6,F.adjust="BH",file="resultsB.txt")
Dr Agnieszka ?mie?ko
Centrum Doskonalosci CENAT
Instytut Chemii Bioorganicznej Polskiej Akademii Nauk
Noskowskiego 12/14
61-704 Pozna?
tel. (61) 8528503 wew. 249
fax: (61) 8520532
Agnieszka Zmienko, Ph.D.
CENAT
Institute of Bioorganic Chemistry
Polish Academy of Sciences
Noskowskiego 12/14
61-704 Poznan, Poland
phone (0048) 61-8528503 ext. 249
fax: (0048) 61-8520532
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