[BioC] Agilent G4112A Arrays
Chuming Chen
chumingchen at gmail.com
Thu Jan 28 14:44:08 CET 2010
Prashantha and everybody comments on my question:
Thank you very much!
Chuming
Prashantha Hebbar wrote:
> Hi Chuming,
>
> I have over looked your previous mail. It seems, there is nothing
> wrong. So, better to follow Wolfgang and Naomi suggestions.
>
> Regards,
> Prashantha
>
> Prashantha Hebbar Kiradi,
> Dept. of Biotechnology,
> Manipal Life Sciences Center,
> Manipal University,
> Manipal, India
> Email:prashantha.hebbar at manipal.edu
>
>
> --- On *Thu, 1/28/10, Chuming Chen /<chumingchen at gmail.com>/* wrote:
>
>
> From: Chuming Chen <chumingchen at gmail.com>
> Subject: Re: [BioC] Agilent G4112A Arrays
> To: "Prashantha Hebbar" <prashantha.hebbar at yahoo.com>
> Cc: bioconductor at stat.math.ethz.ch
> Date: Thursday, January 28, 2010, 4:50 AM
>
> Prashantha and all,
>
> Here is the sessional information regarding my analysis of this
> data set.
>
> Can you point out what I might do wrong?
>
> Thanks,
>
> Chuming
>
> > library(limma)
> >
> > targets <- readTargets("Targets.txt")
> > targets
> SlideNumber Name FileName Cy3 Cy5
> 1 1 B1vsT1 US23502303_251239134396_S02_44k.txt B1 T1
> 2 2 B2vsT2 US23502303_251239134397_S01_44k.txt B2 T2
> 3 3 B3vsT3 US23502303_251239134398_S01_44k.txt B3 T3
> 4 4 B4vsT4 US23502303_251239134399_S01_44k.txt B4 T4
> 5 5 B5vsT5 US23502303_251239134400_S01_44k.txt B5 T5
> >
> > RG <- read.maimages(targets, source="agilent")
> Read US23502303_251239134396_S02_44k.txt
> Read US23502303_251239134397_S01_44k.txt
> Read US23502303_251239134398_S01_44k.txt
> Read US23502303_251239134399_S01_44k.txt
> Read US23502303_251239134400_S01_44k.txt
> >
> > RG <- backgroundCorrect(RG, method="normexp", offset=50)
> Green channel
> Corrected array 1
> Corrected array 2
> Corrected array 3
> Corrected array 4
> Corrected array 5
> Red channel
> Corrected array 1
> Corrected array 2
> Corrected array 3
> Corrected array 4
> Corrected array 5
> >
> > plotDensities(RG)
> >
> > MA <- normalizeBetweenArrays(RG,method="vsn")
> Loading required package: vsn
> Loading required package: Biobase
>
> Welcome to Bioconductor
>
> Vignettes contain introductory material. To view, type
> 'openVignette()'. To cite Bioconductor, see
> 'citation("Biobase")' and for packages 'citation(pkgname)'.
>
> vsn2: 43931 x 10 matrix (1 stratum). Please use 'meanSdPlot' to
> verify the fit.
> >
> > plotDensities(MA)
> >
> > f<-factor(targets$Name)
> > design<-model.matrix(~0+f)
> > design
> fB1vsT1 fB2vsT2 fB3vsT3 fB4vsT4 fB5vsT5
> 1 1 0 0 0 0
> 2 0 1 0 0 0
> 3 0 0 1 0 0
> 4 0 0 0 1 0
> 5 0 0 0 0 1
> attr(,"assign")
> [1] 1 1 1 1 1
> attr(,"contrasts")
> attr(,"contrasts")$f
> [1] "contr.treatment"
>
> > colnames(design) <- levels(f)
> > design
> B1vsT1 B2vsT2 B3vsT3 B4vsT4 B5vsT5
> 1 1 0 0 0 0
> 2 0 1 0 0 0
> 3 0 0 1 0 0
> 4 0 0 0 1 0
> 5 0 0 0 0 1
> attr(,"assign")
> [1] 1 1 1 1 1
> attr(,"contrasts")
> attr(,"contrasts")$f
> [1] "contr.treatment"
>
> >
> > fit<-lmFit(MA, design)
> > contrasts.matrix <- makeContrasts(B1vsT1,B2vsT2, B3vsT3, B4vsT4,
> B5vsT5, levels=design)
> >
> > fit2 <- contrasts.fit(fit, contrasts.matrix)
> > fit2 <- eBayes(fit2)
> Error in ebayes(fit = fit, proportion = proportion, stdev.coef.lim
> = stdev.coef.lim) :
> No residual degrees of freedom in linear model fits
> >
> > toptable(fit2)
> Error in ebayes(fit, ...) :
> No residual degrees of freedom in linear model fits
> >
>
>
> Prashantha Hebbar wrote:
> > Hi Chuming,
> > As per your experimental information, you have replicates.
> Because, you have samples from same tissue with 2 different region
> across all patients. So, you should be able to fit linear model.
> What I guess, there is something wrong in your analysis steps. So,
> better to send sessional information to list.
> > Regards,
> > Prashantha
> >
> > Prashantha Hebbar Kiradi,
> > Dept. of Biotechnology,
> > Manipal Life Sciences Center,
> > Manipal University,
> > Manipal, India
> > Email:prashantha.hebbar at manipal.edu
> >
> > --- On *Mon, 1/25/10, Wolfgang Huber /<whuber at embl.de>/* wrote:
> >
> >
> > From: Wolfgang Huber <whuber at embl.de>
> > Subject: Re: [BioC] Agilent G4112A Arrays
> > To: "Naomi Altman" <naomi at stat.psu.edu>
> > Cc: "Chuming Chen" <chumingchen at gmail.com>, "Prashantha Hebbar"
> > <prashantha.hebbar at yahoo.com>, bioconductor at stat.math.ethz.ch
> > Date: Monday, January 25, 2010, 8:06 PM
> >
> > Hi Chuming
> >
> > if you want to work with the approximation that M-values have
> > equal variances, then preprocessing the data with a method that
> > provides variance stabilisation (e.g. vsn) will likely be
> useful.
> >
> > Furthermore, it might be useful to discard a fraction of genes
> > with low A-values, since they are more likely to be either not
> > expressed, or so weakly expressed that you would find it more
> > difficult to validate them.
> >
> > Best wishes
> > Wolfgang
> >
> > Naomi Altman wrote:
> > > The more data one has, the fewer assumptions one needs.
> In the
> > absence of replication, you cannot get p-values without very
> > strong assumptions. e.g. you could assume that the vast
> majority
> > of the genes do not differentially express, that their M-values
> > have equal variance and that the M-values are normally
> > distributed. Then you could use e.g. the IQR of the M-values to
> > estimate the sd and use this to pick a fold cut-off for DE. You
> > have no reasonable way to estimate FDR with this approach,
> but it
> > might be slightly better than using 2-fold - or then again, it
> > might not. Without replication, there is no way to know.
> > >
> > > Regards,
> > > Naomi Altman
> > >
> > >
> > > At 08:53 AM 1/25/2010, Chuming Chen wrote:
> > >> Hi Prashantha,
> > >>
> > >> Thank you for your suggestion. My target file is as below.
> > Although I couldn't fit a linear model, I still wonder whether I
> > can do some statistic on M (log ratio) values and use the
> p-value
> > to get the differentially expressed genes.
> > >>
> > >> SlideNumber FileName Cy3 Cy5
> > >> 1 B1vsT1.txt B1 T1
> > >> 2 B2vsT2.txt B2 T2
> > >> 3 B3vsT3.txt B3 T3
> > >> 4 B4vsT4.txt B4 T4
> > >> 5 B5vsT5.txt B5 T5
> > >>
> > >> Chuming
> > >>
> > >>
> > >> Prashantha Hebbar wrote:
> > >>> Dear Chen,
> > >>>
> > >>> You need not to look for any other packages. Since, you
> do not
> > have any replicates, do not fit linear model, instead just do
> > normalization with in arrays and look at the M (log ratio)
> values.
> > >>>
> > >>> Regards,
> > >>>
> > >>> Prashantha Hebbar Kiradi,
> > >>> Dept. of Biotechnology,
> > >>> Manipal Life Sciences Center,
> > >>> Manipal University,
> > >>> Manipal, India
> > >>>
> > >>>
> > >>> --- On *Mon, 1/25/10, Chuming Chen /<chumingchen at gmail.com
> >
> <http://us.mc1101.mail.yahoo.com/mc/compose?to=chumingchen@gmail.com>>/*
> > wrote:
> > >>>
> > >>>
> > >>> From: Chuming Chen <chumingchen at gmail.com
> >
> <http://us.mc1101.mail.yahoo.com/mc/compose?to=chumingchen@gmail.com>>
> > >>> Subject: [BioC] Agilent G4112A Arrays
> > >>> To: bioconductor at stat.math.ethz.ch
> >
> <http://us.mc1101.mail.yahoo.com/mc/compose?to=bioconductor@stat.math.ethz.ch>
> > >>> Date: Monday, January 25, 2010, 6:32 AM
> > >>>
> > >>> Dear All,
> > >>>
> > >>> I am trying to find out the differentially expressed
> genes
> > from
> > >>> some Agilent Human Whole Genome (G4112A) Arrays data.
> > >>>
> > >>> I have tried LIMMA package, but LIMMA gave the error
> > message "no
> > >>> residual degrees of freedom in linear model fits" and
> > stopped. My
> > >>> guess is that my data has no replicates in the
> experiment.
> > >>>
> > >>> Is there any other packages I can use to find
> differentially
> > >>> expressed genes which does not require replicates in the
> > experiment?
> > >>>
> > >>> Thanks for your help.
> > >>>
> > >>> Chuming
> > >>>
> > >>> _______________________________________________
> > >>> Bioconductor mailing list
> > >>> Bioconductor at stat.math.ethz.ch
> >
> <http://us.mc1101.mail.yahoo.com/mc/compose?to=Bioconductor@stat.math.ethz.ch>
> > >>> </mc/compose?to=Bioconductor at stat.math.ethz.ch
> >
> <http://us.mc1101.mail.yahoo.com/mc/compose?to=Bioconductor@stat.math.ethz.ch>>
> > >>> https://stat.ethz.ch/mailman/listinfo/bioconductor
> > >>> Search the archives:
> > >>>
> http://news.gmane.org/gmane.science.biology.informatics.conductor
> > >>>
> > >>
> > >> _______________________________________________
> > >> Bioconductor mailing list
> > >> Bioconductor at stat.math.ethz.ch
> >
> <http://us.mc1101.mail.yahoo.com/mc/compose?to=Bioconductor@stat.math.ethz.ch>
> > >> https://stat.ethz.ch/mailman/listinfo/bioconductor
> > >> Search the archives:
> >
> http://news.gmane.org/gmane.science.biology.informatics.conductor
> >
> <http://news..gmane.org/gmane.science.biology.informatics.conductor>
> > >
> > > Naomi S. Altman
> 814-865-3791 (voice)
> > > Associate Professor
> > > Dept. of Statistics
> 814-863-7114 (fax)
> > > Penn State University 814-865-1348
> > (Statistics)
> > > University Park, PA 16802-2111
> > >
> > > _______________________________________________
> > > Bioconductor mailing list
> > > Bioconductor at stat.math.ethz.ch
> >
> <http://us.mc1101.mail.yahoo.com/mc/compose?to=Bioconductor@stat.math.ethz.ch>
> > > https://stat.ethz.ch/mailman/listinfo/bioconductor
> > > Search the archives:
> >
> http://news.gmane.org/gmane.science.biology..informatics.conductor
> >
> <http://news.gmane.org/gmane.science.biology.informatics.conductor>
> >
> > -- Best wishes
> > Wolfgang
> >
> >
> > --
> > Wolfgang Huber
> > EMBL
> > http://www.embl.de/research/units/genome_biology/huber/contact
> >
> >
> >
>
>
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