Entering edit mode
Andreia Fonseca
▴
810
@andreia-fonseca-3796
Last seen 7.2 years ago
Dear list,
I am going to analyze data with the design shown below. I do not have
replicates, but is a complete 2x3 factorial design and therefore I
should be
able to fit a linear model
y=mean+Celltype+Treatment+error for each of gene and then do multiple
testing correction, right? So, why after running,
TS<-paste(raw_files$Cell_Type, raw_files$Treatment, sep=".")
> TS<-factor(TS, levels=c("NS.control1", "NS.H1",
> "NS.H2","S.control2","S.H1","
S.H2"))
> design<-model.matrix(~0+TS)
> contrasts<-makeContrasts(TSS.H1-TSS.H2, TSNS.H1-TSNS.H2,
> (TSNS.H1-TSNS.H2)-(TSS.H1-TSS.H2),levels=design)
> sr.norm2<-sr.norm[order(featureNames(sr.norm)),]
> qDE.limma<-limmaCtData(sr.norm2,design=design,contrasts=contrasts,
> spacing=1)
>
> I am getting an error message
>
> Error in ebayes(fit = fit, proportion = proportion, stdev.coef.lim =
> stdev.coef.lim) :
> No residual degrees of freedom in linear model fits
I get this error?
should I only make a two sample t-test? and I can't fit a linear
model?
Thanks for your help!
Kind regards
Andreia
On Thu, Jan 28, 2010 at 9:54 PM, Heidi Dvinge <heidi@ebi.ac.uk> wrote:
> Hi Andreia,
>
> > Hello Heidi,
> >
> > my question is about the classification of unreliable estimates e
> > setCategory. I saw the code and it seems that you are estimating
just the
> > classical c.i. based in the variation of the data, right?
>
> yep
>
> > I have a new
> > question though concerning qDE.limma, as I told you I have a
factorial
> > design, so I have created a factorial design form the example data
from
> > HT-qPCR
> >
> > TSNS.control1 TSNS.H1 TSNS.H2 TSS.control2 TSS.H1 TSS.H2
> > 1 1 0 0 0 0 0
> > 2 0 1 0 0 0 0
> > 3 0 0 1 0 0 0
> > 4 0 0 0 1 0 0
> > 5 0 0 0 0 1 0
> > 6 0 0 0 0 0 1
> >
> > TS<-paste(raw_files$Cell_Type, raw_files$Treatment, sep=".")
> > TS<-factor(TS, levels=c("NS.control1", "NS.H1",
> > "NS.H2","S.control2","S.H1","S.H2"))
> > design<-model.matrix(~0+TS)
> > contrasts<-makeContrasts(TSS.H1-TSS.H2, TSNS.H1-TSNS.H2,
> > (TSNS.H1-TSNS.H2)-(TSS.H1-TSS.H2),levels=design)
> > sr.norm2<-sr.norm[order(featureNames(sr.norm)),]
> > qDE.limma<-limmaCtData(sr.norm2,design=design,contrasts=contrasts,
> > spacing=1)
> >
> > I am getting an error message
> >
> > Error in ebayes(fit = fit, proportion = proportion, stdev.coef.lim
=
> > stdev.coef.lim) :
> > No residual degrees of freedom in linear model fits
> >
> >
> > is it because this that is not adequate for this design? or I have
the
> > wrong
> > command?
> >
> Your approach as such seems valid, but the problem is that you have
no
> replicate arrays. Which means that unfortunately the limma functions
won't
> work, since there's no way to do statistical testing.
>
> Maybe someone on the list can help if we know a little more about
your
> arrays/intention. It looks e.g. like you don't use your two control
> samples at all?
>
> Cheers
> \Heidi
>
> > thanks Andreia
> >
> >
> >
> >
> > On Wed, Jan 27, 2010 at 11:26 PM, Heidi Dvinge <heidi@ebi.ac.uk>
wrote:
> >
> >> Hello Andreia,
> >>
> >> sorry for the delay in answering. Just to be clear, what
confidence
> >> interval are you referring to? The confidence intervals plotted
in
> >> plotCtOverview? The deviations used to assign categories in then
> >> filtering
> >> the data?
> >>
> >> Cheers
> >> \Heidi
> >>
> >> P.S. By the way, just in case you're new to R, you can always see
the
> >> source code of a function, by just typing the function name in
the
> >> terminal, without "?" before or "()" after.
> >>
> >> > Hi Heidi,
> >> >
> >> >
> >> > sorry is just to say that I have tested to read the file with
two
> >> columns
> >> > for the two factors and that part works!!! So now I will move
on for
> >> the
> >> > other functions, plotCtOverview works fine! Now can you just
answer to
> >> the
> >> > confidence intervals question?
> >> >
> >> > Thanks
> >> > Andreia
> >> > On Wed, Jan 27, 2010 at 10:24 AM, Andreia Fonseca
> >> > <andreia.fonseca@gmail.com> >> >> wrote:
> >> >
> >> >> Hi Heidi,
> >> >>
> >> >>
> >> >> my question is how should be the format of the files.txt file,
so
> >> that
> >> >> HTqPCR can read in the information of the two different
factors,
> >> should
> >> >> it
> >> >> be like the example I wrote below? And what about the
confidence
> >> >> intervals
> >> >> to filter data, how does the package estimates them?
> >> >> Cheers
> >> >> Andreia
> >> >>
> >> >>
> >> >> On Tue, Jan 26, 2010 at 10:23 PM, Heidi Dvinge
<heidi@ebi.ac.uk>
> >> wrote:
> >> >>
> >> >>> Hello Andreia,
> >> >>>
> >> >>> actually, HTqPCR can handle multi-factor design, there's just
no
> >> >>> example
> >> >>> of that in the vignette (will consider adding it in the next
> >> revision).
> >> >>>
> >> >>> The function limmaCtData takes all the arguments that you'd
use if
> >> you
> >> >>> were analysing microarray data using lmFit and contrasts.fit
from
> >> the
> >> >>> limma package. You need to specify the design and contrast
matrix
> >> >>> yourself though. As I recall, the limma user's guide has a
couple of
> >> >>> example involving factorial design.
> >> >>>
> >> >>> HTH
> >> >>> \Heidi
> >> >>>
> >> >>> > Dear list,
> >> >>> >
> >> >>> > Soon I will receive data from the qpcr Exicon platform to
analyze
> >> and
> >> >>> I
> >> >>> > have
> >> >>> > been playing around with HTqPCR package, however from the
vignete,
> >> it
> >> >>> > seems
> >> >>> > that is only capable of deleting with data design of one
factor,
> >> how
> >> >>> do
> >> >>> I
> >> >>> > handle it with a factorial design, namely how do I read the
data
> >> and
> >> >>> > create
> >> >>> > the model.matrix, the examples only consider one factor.
Another
> >> >>> question
> >> >>> > is
> >> >>> > concerning part 5 of the vignete, how are the Confidence
values
> >> >>> estimated?
> >> >>> > is is based in the variance of the data?
> >> >>> >
> >> >>> > In order for you to understand my doubts here is my design
is a
> >> 2x3
> >> >>> > design,
> >> >>> > I don't have replicates, but each file has the CT values
for each
> >> >>> gene
> >> >>> > frome
> >> >>> > pooled RNA of 10 patients.
> >> >>> > factor1 factor2
> >> >>> > file1.txt NS C
> >> >>> > file2.txt NS H1
> >> >>> > file3.txt NS H2
> >> >>> > file4.txt S C
> >> >>> > file5.txt S H1
> >> >>> > file6.txt S H2
> >> >>> >
> >> >>> >
> >> >>> > Thanks for your help.
> >> >>> > with kind regards,
> >> >>> > Andreia
> >> >>> >
> >> >>> >
> >> >>> > --
> >> >>> > --------------------------------------------
> >> >>> > Andreia J. Amaral
> >> >>> > Unidade de Imunologia Clínica
> >> >>> > Instituto de Medicina Molecular
> >> >>> > Universidade de Lisboa
> >> >>> > email: andreiaamaral@fm.ul.pt
> >> >>> > andreia.fonseca@gmail.com
> >> >>> >
> >> >>> > [[alternative HTML version deleted]]
> >> >>> >
> >> >>> > _______________________________________________
> >> >>> > Bioconductor mailing list
> >> >>> > Bioconductor@stat.math.ethz.ch
> >> >>> > https://stat.ethz.ch/mailman/listinfo/bioconductor
> >> >>> > Search the archives:
> >> >>> >
http://news.gmane.org/gmane.science.biology.informatics.conductor
> >> >>>
> >> >>>
> >> >>>
> >> >>
> >> >>
> >> >> --
> >> >> --------------------------------------------
> >> >> Andreia J. Amaral
> >> >> Unidade de Imunologia Clínica
> >> >> Instituto de Medicina Molecular
> >> >> Universidade de Lisboa
> >> >> email: andreiaamaral@fm.ul.pt
> >> >> andreia.fonseca@gmail.com
> >> >>
> >> >
> >> >
> >> >
> >> > --
> >> > --------------------------------------------
> >> > Andreia J. Amaral
> >> > Unidade de Imunologia Clínica
> >> > Instituto de Medicina Molecular
> >> > Universidade de Lisboa
> >> > email: andreiaamaral@fm.ul.pt
> >> > andreia.fonseca@gmail.com
> >> >
> >>
> >>
> >>
> >
> >
> > --
> > --------------------------------------------
> > Andreia J. Amaral
> > Unidade de Imunologia Clínica
> > Instituto de Medicina Molecular
> > Universidade de Lisboa
> > email: andreiaamaral@fm.ul.pt
> > andreia.fonseca@gmail.com
> >
>
>
>
--
--------------------------------------------
Andreia J. Amaral
Unidade de Imunologia Clínica
Instituto de Medicina Molecular
Universidade de Lisboa
email: andreiaamaral@fm.ul.pt
andreia.fonseca@gmail.com
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