[BioC] limma - batch dates incorrporated into t-test
Helen Smith
helen.smith-2 at manchester.ac.uk
Thu Sep 12 17:50:44 CEST 2013
That seems to have all worked perfectly thank you so much!
Just to double check: my batch dates aren't a contrast but have been incorporated into the t-test as a factor using the following code after you contrast line:
###Contrast matrix###
contrast <- makeContrasts(oocyte - four_cell,oocyte - eight_cell,oocyte - blastomere,oocyte - blastocyst,four_cell - eight_cell,four_cell - blastomere,four_cell - blastocyst,eight_cell - blastomere,eight_cell - blastocyst,blastomere - blastocyst, levels =design)
###Fit linear model###
fit <- lmFit(eset, design)
names(fit)
fit2 <- contrasts.fit(fit, contrast)
fit2 <- eBayes(fit2)
write.csv(fit2,"Limma_fit2.csv")
Thanks again,
Helen
-----Original Message-----
From: James W. MacDonald [mailto:jmacdon at uw.edu]
Sent: 12 September 2013 16:16
To: Helen Smith
Cc: bioconductor at r-project.org
Subject: Re: [BioC] limma - batch dates incorrporated into t-test
Ugh, tripped up by syntactically invalid R names! That's why I removed those nasty numbers from 4cell and 8cell, but still and yet got tripped up...
So the dates are what we call 'nuisance variables', which are things you want to account for, but have no real interest for you. Because of this we can call them anything we want, so long as R will accept them.
And what R wants is something that doesn't start with a number, and doesn't have math symbols in it (which is the problem with e.g., Date02/07/2013).
And I think there is a problem with the gsub() line to get rid of the prepended cruft that R likes to add to model.matrix colnames. So how about
colnames(design) <- gsub("Target", "", colnames(design))
then you need to change the colnames that have Date in them as well.
These should be columns 5-11. So you can just do
colnames(design)[5:11] <- paste0("Date", 1:7) ## that's paste with a Zero, not capital O
You should check the colnames just to make sure I didn't count wrong.
And you should probably smash your computer against the wall anyway.
They make such a pretty tinkly noise when they shatter. ;-D
Best,
Jim
On Thursday, September 12, 2013 10:26:55 AM, Helen Smith wrote:
> Thanks James, that all worked great apart from the last line:
>
> contrast <- makeContrasts(blastomere - oocyte, blastomere -
> eight_cell, blastomere - four_cell, <added all my contrasts here>,
> levels =design)
>
> Which produced the following error:
>
> /Error in makeContrasts(oocyte - four_cell, oocyte - eight_cell,
> oocyte - : /
>
> / The levels must by syntactically valid names in R, see
> help(make.names). Non-valid names:
> Date02/07/2013,Date06/03/2013,Date10/01/2008,Date13/02/2007,Date16/02/
> 2007,Date23/03/2007,Date24/10/2007/
>
> //
>
> Ideas?
>
> Thank you, you have turned a frustrating day into one in which I can
> see light at the end of the tunnel…….
>
> -----Original Message-----
> From: James W. MacDonald [mailto:jmacdon at uw.edu]
> Sent: 12 September 2013 15:01
> To: Helen Smith
> Cc: bioconductor at r-project.org
> Subject: Re: [BioC] limma - batch dates incorrporated into t-test
>
> Hi Helen,
>
> On Thursday, September 12, 2013 9:02:25 AM, Helen Smith wrote:
>
> > Hi All,
>
> >
>
> > I am new to limma and R programming.
>
> >
>
> > I have the CEL.files uploaded, normalised as an expression set, and
> have put together the target.txt file containing the following layout:
>
> > Name FileName Target Date
>
> > 5 0207_DaB7_H_ea_Blast1.CEL blastocyst
> 13/02/2007
>
> > 6 0207_DaB8_H_ea_Blast2.CEL blastocyst
> 13/02/2007
>
> > 4 0207_DaB6_H_ea_4cell_r3.CEL 4cell 16/02/2007
>
> > 16 0307_DaB1_H_ea_oocyte_r1.CEL oocyte 23/03/2007
>
> > 17 0307_DaB3_H_ea_oocyte_r3.CEL oocyte 23/03/2007
>
> > 18 0307_DaB5_H_ea_4cell_r2.CEL 4cell 23/03/2007
>
> > 21 1007_DaB4_H_ea_4cell_r1.CEL 4cell 24/10/2007
>
> > 22 1007_DaB9_H_ea_Blast3.CEL blastocyst
> 24/10/2007
>
> > 23 1007_DaB21_H_ea_oocyte9856.CEL oocyte 24/10/2007
>
> > 24 1007_DaB22_H_ea_oocyte9858.CEL oocyte 24/10/2007
>
> > 1 0108_DaB18_H_ea_4cell_0.9_r4.CEL 4cell
> 10/01/2008
>
> > 2 0108_DaB20_H_ea_4cell_0.9_r6.CEL 4cell
> 10/01/2008
>
> > 3 0108_DaB23_H_ea_Blast_2.5_r4.CEL
> blastocyst 10/01/2008
>
> > 10 0213_SueK(2)4_H_ea_005_B4.CEL
> blastomere 01/03/2013
>
> > 14 0213_SueK(2)8_H_ea_005_B8.CEL
> blastomere 01/03/2013
>
> > 15 0213_SueK(2)10_H_ea_002_8CE.CEL 8cell 01/03/2013
>
> > 7 0213_SueK(2)1_H_ea_005_B1.CEL
> blastomere 06/03/2013
>
> > 8 0213_SueK(2)2_H_ea_005_B2.CEL
> blastomere 06/03/2013
>
> > 9 0213_SueK(2)3_H_ea_005_B3.CEL
> blastomere 06/03/2013
>
> > 11 0213_SueK(2)5_H_ea_005_B5.CEL
> blastomere 06/03/2013
>
> > 12 0213_SueK(2)6_H_ea_005_B6.CEL
> blastomere 06/03/2013
>
> > 13 0213_SueK(2)7_H_ea_005_B7.CEL
> blastomere 06/03/2013
>
> > 19 0713_Suek(3)2_Ep_H_8C_006_11.CEL 8cell 02/07/2013
>
> > 20 0713_Suek(3)3_Ep_H_8C_004_11.CEL 8cell 02/07/2013
>
> >
>
> >
>
> > But am struggling with setting up the design matrix and contrasts.
> > My
>
> > limited programming ability is probably playing the largest role,
> > and
>
> > I feel like smashing the computer against the wall (probably a sign
> > of
>
> > slight frustration ïŠ)
>
> It's really quite simple ;-D. You just make a design matrix that
> includes the dates, and then make a contrasts matrix that doesn't use
> the dates to make comparisons.
>
> The simplest way to do this is to use model.matrix() and makeContrasts().
>
> Assume you read in your targets.txt file like so:
>
> targets <- read.table("targets.txt", header=TRUE, stringsAsFactors =
>
> FALSE)
>
> we have to do the stringsAsFactors business because your 4cell and
> 8cell sample designations won't work with makeContrasts() (they cannot
> start with a number).
>
> targets$Targets <- factor(gsub("4", "four_", gsub("8", "eight_",
>
> targets$Targets)))
>
> then
>
> design <- model.matrix(~0+Target+Date, targets)
>
> Now you probably have column names with an extra 'targets' in the
> name, which nobody likes, so we remove
>
> colnames(design) <- gsub("targets", "", colnames(design))
>
> contrast <- makeContrasts(blastomere - oocyte, blastomere -
> eight_cell, blastomere - four_cell, <add all the contrasts you want
> here>, levels =
>
> design)
>
> and now you proceed on to the lmFit/contrasts.fit/eBayes steps.
>
> Best,
>
> Jim
>
> >
>
> > The batch dates are included in the Target file, as I would like to
>
> > add them as a factor when computing the T-test. But this isn’t a
>
> > contrast I would like to make on its own (does that make sense?)
>
> >
>
> > I would like to make all possible combinations of contrasts, listed
> as targets.
>
> >
>
> > Any help would be fantastic!!!!!
>
> > Thank you in advance,
>
> > Helen
>
> >
>
> >
>
> >
>
> >
>
> > [[alternative HTML version deleted]]
>
> >
>
> >
>
> >
>
> > _______________________________________________
>
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>
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>
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>
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>
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>
> --
>
> James W. MacDonald, M.S.
>
> Biostatistician
>
> University of Washington
>
> Environmental and Occupational Health Sciences
>
> 4225 Roosevelt Way NE, # 100
>
> Seattle WA 98105-6099
>
--
James W. MacDonald, M.S.
Biostatistician
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099
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