Hi All,
I have a complex experiment (for me) and I do not known how do I do to
normalize it.
More specifically, I don't know as building the file samples (targets)
for marray.
The design is:
Time
1day 2day 3day
Rep1 Rep1 Rep1
Un Treated Rep2 Rep2 Rep2
Rep3 Rep3 Rep3
Rep1 Rep1 Rep1
Un Treated Rep2 Rep2 Rep2
Rep3 Rep3 Rep3
If I have one time, my targets file for marrayinput is like this:
# of slide Names experiment Cy3 experiment Cy5 date
1 File1 Un Treated Treated 19/01/2004
It is a temporary series with three different times and three
repetitions in each one of the times.
Me already analysed some simpler experiments. For example, I know to
analyse inside of every time, individually. However, I didn't get to
find an example alike to mine in the marray vignettes.
After the normalization, I am thinking about using limma.
I would like to know which genes were differentialy expressed in every
time. Besides, would I like to verify the behavior of these genes
along the time (for example, were they increased or done decreased
along the time?). I already had looking at the limma user's guide and
I saw that there is the function heatdiagram.
I will need to analyze it in the marray in a way that is easier of
being analyzed in limma. Another doubt that I already have on limma
would be the file design.
All help will be very welcome.
Best wishes.
--
Marcelo Luiz de Laia, M.Sc.
Dep. de Tecnologia, Lab. Bioqu?mica e de Biologia Molecular
Universidade Estadual Paulista - UNESP
Via de Acesso Prof. Paulo Donato Castelane, Km 05
14.884-900 - Jaboticabal, SP, Brazil
PhoneFax: 16 3209-2675/2676/2677 R. 202/208/203 (trab.)
HomePhone: 16 3203 2328 - www.lbm.fcav.unesp.br - mlaia@yahoo.com
At 12:52 AM 20/01/2004, Marcelo Luiz de Laia wrote:
>Hi All,
>
>I have a complex experiment (for me) and I do not known how do I do
to
>normalize it.
Why not normalize it exactly has you've normalized data in earlier
studies?
>More specifically, I don't know as building the file samples
(targets) for
>marray.
>
>The design is:
>
> Time
> 1day 2day 3day
>
> Rep1 Rep1 Rep1
>Un Treated Rep2 Rep2 Rep2
> Rep3 Rep3 Rep3
>
> Rep1 Rep1 Rep1
>Un Treated Rep2 Rep2 Rep2
> Rep3 Rep3 Rep3
>
>If I have one time, my targets file for marrayinput is like this:
>
># of slide Names experiment Cy3 experiment Cy5 date
>1 File1 Un Treated Treated 19/01/2004
>
>It is a temporary series with three different times and three
repetitions
>in each one of the times.
>
>Me already analysed some simpler experiments. For example, I know to
>analyse inside of every time, individually. However, I didn't get to
find
>an example alike to mine in the marray vignettes.
>
>After the normalization, I am thinking about using limma.
>
>I would like to know which genes were differentialy expressed in
every
>time. Besides, would I like to verify the behavior of these genes
along
>the time (for example, were they increased or done decreased along
the
>time?). I already had looking at the limma user's guide and I saw
that
>there is the function heatdiagram.
Heatdiagram may help you visualize your results, but what you really
need
is the F-statistic computed by the classifyTests() function. This is
not
yet explained in the User's Guide. Can you consult a local
statistician for
help who knows a little about linear models and contrasts?
Gordon
>I will need to analyze it in the marray in a way that is easier of
being
>analyzed in limma. Another doubt that I already have on limma would
be the
>file design.
>
>All help will be very welcome.
>
>Best wishes.
>
>--
>Marcelo Luiz de Laia, M.Sc.
>Dep. de Tecnologia, Lab. Bioqu?mica e de Biologia Molecular
>Universidade Estadual Paulista - UNESP
>Via de Acesso Prof. Paulo Donato Castelane, Km 05
>14.884-900 - Jaboticabal, SP, Brazil
>PhoneFax: 16 3209-2675/2676/2677 R. 202/208/203 (trab.)
>HomePhone: 16 3203 2328 - www.lbm.fcav.unesp.br - mlaia@yahoo.com
Dear Gordon and All,
In my laboratory is impossible to have a statistician involved in
anlaysis, now.
My doubt about the normalization with marray appeared due I to have to
use limma, after. Then, I thought: Maybe there be a way to enter with
the data in marray so that the analyses in limma are easier.
My questions, basically, are:
- Which genes are up-regulated in the three times?
- Which genes are down-regulated in the three times?
- Which are up-regulateds in the time 1 and later they do decrease in
the times 2 and 3?
- Which are up-regulateds in the times 1 and 2 and later it does
decrease in the time 3?
- Which are down-regulateds in the time 1 and up-regulated in the
times 2 and 3?
- Which are down-regulateds in the times 1 and 2 and up-regulated in
the time 3?
I believe that these are the main subjects. Would you have
suggestions?
I hope to analyze our data with the help of the members of the list
Bioconductor.
Thanks a lot for your interest in help me.
The experiment design is:
Time
1day 2day 3day
Rep1 Rep1 Rep1
Un Treated Rep2 Rep2 Rep2
Rep3 Rep3 Rep3
Rep1 Rep1 Rep1
Treated Rep2 Rep2 Rep2
Rep3 Rep3 Rep3
Marcelo
Em Tue, 20 Jan 2004 10:42:40 +1100
Gordon Smyth <smyth@wehi.edu.au> escreveu:
GS> At 12:52 AM 20/01/2004, Marcelo Luiz de Laia wrote:
GS> >Hi All,
GS> >
GS> >I have a complex experiment (for me) and I do not known how do I
do to
GS> >normalize it.
GS>
GS> Why not normalize it exactly has you've normalized data in earlier
studies?
GS>
GS> >More specifically, I don't know as building the file samples
(targets) for
GS> >marray.
GS> >
GS> >The design is:
GS> >
GS> > Time
GS> > 1day 2day 3day
GS> >
GS> > Rep1 Rep1 Rep1
GS> >Un Treated Rep2 Rep2 Rep2
GS> > Rep3 Rep3 Rep3
GS> >
GS> > Rep1 Rep1 Rep1
GS> >Un Treated Rep2 Rep2 Rep2
GS> > Rep3 Rep3 Rep3
GS> >
GS> >If I have one time, my targets file for marrayinput is like this:
GS> >
GS> ># of slide Names experiment Cy3 experiment Cy5 date
GS> >1 File1 Un Treated Treated 19/01/2004
GS> >
GS> >It is a temporary series with three different times and three
repetitions
GS> >in each one of the times.
GS> >
GS> >Me already analysed some simpler experiments. For example, I know
to
GS> >analyse inside of every time, individually. However, I didn't get
to find
GS> >an example alike to mine in the marray vignettes.
GS> >
GS> >After the normalization, I am thinking about using limma.
GS> >
GS> >I would like to know which genes were differentialy expressed in
every
GS> >time. Besides, would I like to verify the behavior of these genes
along
GS> >the time (for example, were they increased or done decreased
along the
GS> >time?). I already had looking at the limma user's guide and I saw
that
GS> >there is the function heatdiagram.
GS>
GS> Heatdiagram may help you visualize your results, but what you
really need
GS> is the F-statistic computed by the classifyTests() function. This
is not
GS> yet explained in the User's Guide. Can you consult a local
statistician for
GS> help who knows a little about linear models and contrasts?
GS>
GS> Gordon
GS>
GS> >I will need to analyze it in the marray in a way that is easier
of being
GS> >analyzed in limma. Another doubt that I already have on limma
would be the
GS> >file design.
GS> >
GS> >All help will be very welcome.
GS> >
GS> >Best wishes.
GS> >
GS> >--
GS> >Marcelo Luiz de Laia, M.Sc.
GS> >Dep. de Tecnologia, Lab. Bioqu?mica e de Biologia Molecular
GS> >Universidade Estadual Paulista - UNESP
GS> >Via de Acesso Prof. Paulo Donato Castelane, Km 05
GS> >14.884-900 - Jaboticabal, SP, Brazil
GS> >PhoneFax: 16 3209-2675/2676/2677 R. 202/208/203 (trab.)
GS> >HomePhone: 16 3203 2328 - www.lbm.fcav.unesp.br - mlaia@yahoo.com
GS>
--
Marcelo Luiz de Laia, M.Sc.
Dep. de Tecnologia, Lab. Bioqu?mica e de Biologia Molecular
Universidade Estadual Paulista - UNESP
Via de Acesso Prof. Paulo Donato Castelane, Km 05
14.884-900 - Jaboticabal, SP, Brazil
PhoneFax: 16 3209-2675/2676/2677 R. 202/208/203 (trab.)
HomePhone: 16 3203 2328 - www.lbm.fcav.unesp.br - mlaia@yahoo.com
At 11:20 PM 20/01/2004, Marcelo Luiz de Laia wrote:
>Dear Gordon and All,
>
>In my laboratory is impossible to have a statistician involved
>in anlaysis, now.
>
>My doubt about the normalization with marray appeared due I to have
to use
>limma, after. Then, I thought: Maybe there be a way to enter with the
data
>in marray so that the analyses in limma are easier.
You can enter your data in marraInput or using limma. It makes no
difference for the analysis. It's simply a matter of what you find
easiest
to use.
Gordon
>My questions, basically, are:
>- Which genes are up-regulated in the three times?
>- Which genes are down-regulated in the three times?
>- Which are up-regulateds in the time 1 and later they do decrease in
the
>times 2 and 3?
>- Which are up-regulateds in the times 1 and 2 and later it does
decrease
>in the time 3?
>- Which are down-regulateds in the time 1 and up-regulated in the
times 2
>and 3?
>- Which are down-regulateds in the times 1 and 2 and up-regulated in
the
>time 3?
>I believe that these are the main subjects. Would you have
suggestions?
>
>I hope to analyze our data with the help of the members of the list
>Bioconductor.
>
>Thanks a lot for your interest in help me.
>
>The experiment design is:
>
> Time
> 1day 2day 3day
>
> Rep1 Rep1 Rep1
>Un Treated Rep2 Rep2 Rep2
> Rep3 Rep3 Rep3
>
> Rep1 Rep1 Rep1
> Treated Rep2 Rep2 Rep2
> Rep3 Rep3 Rep3
>
>Marcelo
>
>
>Em Tue, 20 Jan 2004 10:42:40 +1100
>Gordon Smyth <smyth@wehi.edu.au> escreveu:
>
>GS> At 12:52 AM 20/01/2004, Marcelo Luiz de Laia wrote:
>GS> >Hi All,
>GS> >
>GS> >I have a complex experiment (for me) and I do not known how do I
do to
>GS> >normalize it.
>GS>
>GS> Why not normalize it exactly has you've normalized data in
earlier
>studies?
>GS>
>GS> >More specifically, I don't know as building the file samples
>(targets) for
>GS> >marray.
>GS> >
>GS> >The design is:
>GS> >
>GS> > Time
>GS> > 1day 2day 3day
>GS> >
>GS> > Rep1 Rep1 Rep1
>GS> >Un Treated Rep2 Rep2 Rep2
>GS> > Rep3 Rep3 Rep3
>GS> >
>GS> > Rep1 Rep1 Rep1
>GS> >Un Treated Rep2 Rep2 Rep2
>GS> > Rep3 Rep3 Rep3
>GS> >
>GS> >If I have one time, my targets file for marrayinput is like
this:
>GS> >
>GS> ># of slide Names experiment Cy3 experiment Cy5 date
>GS> >1 File1 Un Treated Treated 19/01/2004
>GS> >
>GS> >It is a temporary series with three different times and three
>repetitions
>GS> >in each one of the times.
>GS> >
>GS> >Me already analysed some simpler experiments. For example, I
know to
>GS> >analyse inside of every time, individually. However, I didn't
get to
>find
>GS> >an example alike to mine in the marray vignettes.
>GS> >
>GS> >After the normalization, I am thinking about using limma.
>GS> >
>GS> >I would like to know which genes were differentialy expressed in
every
>GS> >time. Besides, would I like to verify the behavior of these
genes along
>GS> >the time (for example, were they increased or done decreased
along the
>GS> >time?). I already had looking at the limma user's guide and I
saw that
>GS> >there is the function heatdiagram.
>GS>
>GS> Heatdiagram may help you visualize your results, but what you
really need
>GS> is the F-statistic computed by the classifyTests() function. This
is not
>GS> yet explained in the User's Guide. Can you consult a local
>statistician for
>GS> help who knows a little about linear models and contrasts?
>GS>
>GS> Gordon
>GS>
>GS> >I will need to analyze it in the marray in a way that is easier
of being
>GS> >analyzed in limma. Another doubt that I already have on limma
would
>be the
>GS> >file design.
>GS> >
>GS> >All help will be very welcome.
>GS> >
>GS> >Best wishes.
>GS> >
>GS> >--
>GS> >Marcelo Luiz de Laia, M.Sc.
>GS> >Dep. de Tecnologia, Lab. Bioqu?mica e de Biologia Molecular
>GS> >Universidade Estadual Paulista - UNESP
>GS> >Via de Acesso Prof. Paulo Donato Castelane, Km 05
>GS> >14.884-900 - Jaboticabal, SP, Brazil
>GS> >PhoneFax: 16 3209-2675/2676/2677 R. 202/208/203 (trab.)
>GS> >HomePhone: 16 3203 2328 - www.lbm.fcav.unesp.br -
mlaia@yahoo.com
>GS>
>
>
>--
>Marcelo Luiz de Laia, M.Sc.
>Dep. de Tecnologia, Lab. Bioqu?mica e de Biologia Molecular
>Universidade Estadual Paulista - UNESP
>Via de Acesso Prof. Paulo Donato Castelane, Km 05
>14.884-900 - Jaboticabal, SP, Brazil
>PhoneFax: 16 3209-2675/2676/2677 R. 202/208/203 (trab.)
>HomePhone: 16 3203 2328 - www.lbm.fcav.unesp.br - mlaia@yahoo.com
In the Tue, 20 Jan 2004 10:42:40 +1100
Gordon Smyth <smyth@wehi.edu.au> write:
GS> Heatdiagram may help you visualize your results, but what you
really need
GS> is the F-statistic computed by the classifyTests() function. This
is not
GS> yet explained in the User's Guide. Can you consult a local
statistician for
GS> help who knows a little about linear models and contrasts?
GS>
GS> Gordon
Hi Gordon and All,
I find in the html_help and in the user's guide and I accomplished
the following test with my data.
If is possible, I would like you to verify if it is correct, because
I am not sure of that.
The experiment design is:
Time
1day 2day 3day
Rep1 Rep1 Rep1
Un Treated Rep2 Rep2 Rep2
Rep3 Rep3 Rep3
Rep1 Rep1 Rep1
Treated Rep2 Rep2 Rep2
Rep3 Rep3 Rep3
It follows the script:
> library(limma)
> RG <- read.maimages(files, columns=list(Rf="DataVol",Gf="CtrlVol",
+ Rb="DataBkgd",Gb="CtrlBkgd"))
> show(RG)
> summary(RG$R)
> genes.names[1:10,]
> printer <- list(ngrid.r=4, ngrid.c=5, nspot.r=16, nspot.c=24,
ndups=2,
+ spacing=1, npins=20, start="topleft")
> printer
> MA <- normalizeWithinArrays(RG, method="none", printer)
> boxplot(MA$M~col(MA$M))
> MA <- normalizeWithinArrays(RG, printer)
> boxplot(MA$M~col(MA$M))
> MA.fa <- normalizeBetweenArrays(MA,method="scale")
> boxplot(MA.fa$M~col(MA.fa$M))
> design <- model.matrix(~ -1+factor(c(1,1,1,2,2,2,3,3,3)))
> colnames(design) <- c("time1","time2","time3")
> fit <- lmFit(MA.fa,design)
> contrast.matrix <- makeContrasts(time2-time1,
time3-time2,time3-time1,levels=design)
> fit2 <- contrasts.fit(fit,contrast.matrix)
> fit3 <- eBayes(fit2)
> time2.time1 <- topTable(fit3, coef=1, adjust="fdr")
> time3.time2 <- topTable(fit3, coef=2, adjust="fdr")
> time3.time1 <- topTable(fit3, coef=3, adjust="fdr")
> clas <- classifyTests(fit3)
> vennDiagram(clas)
If the script is correct, I obtained genes common to the time 2 and
time 1, time 3 and time 2 and time 3 and time 1. However, I didn't
obtain any gene common to the three times.
Is there as leaving the test a little less rigorous?
Maybe I find at least about 5 genes common to the three times! Or it
is not
a good ideia?
Thanks very much
Best wishes
Marcelo
GS>
