Entering edit mode
Stephen Moore
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70
@stephen-moore-492
Last seen 10.3 years ago
Hi Fai,
I must point out that I am not a statistician, however, I think your
thinking is correct. If you pool samples and then hybridize that one
sample to three different chips, then you would be assessing the
variation between the Affy platforms rather than allowing for
variation in the different biological samples and since the variation
between the Affy platforms (as I understand it)tends to be small it
may not be worthwhile doing this. However if you make three pooled
samples and hyb to three different chips and then use the average of
the three for your reference then I think you will be getting a better
representation of biological variability.
Cheers
Steve.
-----Original Message-----
From: bioconductor-request@stat.math.ethz.ch
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Sent: 20 January 2004 22:49
To: bioconductor@stat.math.ethz.ch
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Today's Topics:
1. Re: [maNorm] Normalization a complex experiment...
(Marcelo Luiz de Laia)
2. limma and makeContrasts (Stephen Henderson)
3. Re: Blurriness assesment of scanner TIFF files (Simon Lin)
4. RE: [maImage: Draw multiple spatial plots on the SAME grap h]
(Naomi Altman)
5. limma: out of bounds error (Straubhaar, Juerg)
6. Trouble installing 'makecdfenv' (Jim Breaux)
7. Proper pooling design (YUK FAI LEUNG)
----------------------------------------------------------------------
Message: 1
Date: Tue, 20 Jan 2004 09:20:34 -0300
From: Marcelo Luiz de Laia <mlaia@fcav.unesp.br>
Subject: Re: [BioC] [maNorm] Normalization a complex experiment...
To: bioconductor@stat.math.ethz.ch
Message-ID: <20040120092034.00002c10@lbmsala4>
Content-Type: text/plain; charset=ISO-8859-15
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
------------------------------
Message: 2
Date: Tue, 20 Jan 2004 12:38:11 -0000
From: Stephen Henderson <s.henderson@ucl.ac.uk>
Subject: [BioC] limma and makeContrasts
To: "'Bioconductor@stat.math.ethz.ch'"
<bioconductor@stat.math.ethz.ch>
Message-ID:
<e7cf6bc2744cbe41ae4e87635c7c893c1c668e@exc.wibr.ucl.ac.uk>
Content-Type: text/plain
Hi
I've been using the limma and eBayes function for looking at a set of
affymetrix chips with 8 or so different groups within. Following the
instructions on page 11=12 of the PDF guide, this works fine I think.
My question regards different ways to specify the contrasts. If I wish
to
compare composite groups, e.g.
(group1,group2) vs (group3,group4) and others
is there a syntax to specify this within the makeContrasts function?,
and
where can I read about this?, or alternatively is there a better step
at
which I can achieve this (without changing my class vector repeatedly
that
is)?
Stephen
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This email and any files transmitted with it are
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------------------------------
Message: 3
Date: Tue, 20 Jan 2004 09:42:34 -0500
From: "Simon Lin" <simon.lin@duke.edu>
Subject: [BioC] Re: Blurriness assesment of scanner TIFF files
To: <bioconductor@stat.math.ethz.ch>
Message-ID: <002201c3df63$a46366c0$71761098@ccis1184>
Content-Type: text/plain; charset="iso-8859-1"
Hi, Edo:
I would like to share some opinions slightly outside of the image
analysis
context.
To calibrate the scanner, it is better to use some calibration slides.
There
should be some 'standard patterns' on this calibration slide. Like the
'test
paper' used by the Xerox copier repairman with lines and meshes at
different
intervals, or the checker board pattern used by the TV repairman.
To make this calibration slide, a high-end solution is photo-etching.
A
quick solution is to spot some fluorescent dyes at known positions.
Even for people with no obvious problem of the scanner, this should be
checked at a regular interval to ensure the operating condition of the
scanner. Ask the scanner maker; they may do the service.
An accurate physical measurement will solve many of our headaches down
the
road!
Good luck!
Simon
=================================================
Simon M. Lin, M.D.
Manager, Duke Bioinformatics Shared Resource
Assistant Research Professor, Biostatistics and Bioinformatics
Box 3958, Duke University Medical Center
Durham, NC 27710
Ph: (919) 681-9646 FAX: (919) 681-8028
Lin00025 (at) mc.duke.edu
http://dbsr.duke.edu
=================================================
Message: 3
Date: Mon, 19 Jan 2004 16:38:55 +0100
From: "Edo Plantinga" <a.e.d.plantinga@med.rug.nl>
Subject: [BioC] Blurriness assesment of scanner TIFF files
To: "Bioconductor" <bioconductor@stat.math.ethz.ch>
Message-ID: <002801c3dea2$59154420$a38f7d81@edo>
Content-Type: text/plain
Dear all,
At our department we have experienced some difficulties with our
microarray
scanner. I am looking for some software that can read in the raw TIFF
files
that come out of our scanner to asses how blurry the picture is (i.e.
how
sharp the edges are in the picture). I would also like to know which
areas
of the picture are the most blurry (we suspect a left to right
effect). Does
anyone know of (R?) software that can do this?
Kind regards,
Edo Plantinga
[[alternative HTML version deleted]]
------------------------------
Message: 4
Date: Tue, 20 Jan 2004 09:43:37 -0500
From: Naomi Altman <naomi@stat.psu.edu>
Subject: RE: [BioC] [maImage: Draw multiple spatial plots on the SAME
grap h]
To: "Foata,Francis,LAUSANNE,NRC/N&H" <francis.foata@rdls.nestle.com>
Cc: bioconductor@stat.math.ethz.ch
Message-ID: <6.0.0.22.2.20040120093920.01df5b40@stat.psu.edu>
Content-Type: text/plain; charset="us-ascii"
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------------------------------
Message: 5
Date: Tue, 20 Jan 2004 17:06:25 -0500
From: "Straubhaar, Juerg" <juerg.straubhaar@umassmed.edu>
Subject: [BioC] limma: out of bounds error
To: <bioconductor@stat.math.ethz.ch>
Message-ID:
<1A42F1E1A1E73A4F8C6048789F34A32F2D8A99@edunivmail02.ad.umassmed.edu>
Content-Type: text/plain; charset="Windows-1252"
Dear Dr. Smyth,
I am analysing a series of two-colour microarray data sets with limma.
The sets were downloaded from SMD (Standford Microarray Database) and
I read the data with the command:
targets <- readTargets('N20targets.txt')
RG<-read.maimages(targets$FileName, source="smd", fill=T,
wt.fun=function(x) {return(x$FLAG)})
After reading the gal file and layout I proceeded with the
normalization:
MA<-normalizeWithinArrays(RG)
This function terminated prematurely with an 'out of bounds' error. I
found the error in the printtiploess code block of the
normalizeWithinArrays function. The layout with 8 X 4 print grids,
each containing 650 spots, provides for 20800 spots. The chips I am
using have 20736 spots. I added a small amount of code to your
normalizeWithinArrays() which eliminated the error. The code I added
is (after #comment)
printtiploess = {^M
if(is.null(layout)) stop("Layout argument not
specified")^M
ngr <- layout$ngrid.r^M
ngc <- layout$ngrid.c^M
nspots <- layout$nspot.r * layout$nspot.c^M
for (j in 1:narrays) {^M
spots <- 1:nspots^M
for (gridr in 1:ngr)^M
for (gridc in 1:ngc) {^M
# modified: SMD data files smaller than ngr * ngc * spots!^M
if(spots[nspots] > nrow(object$M)) {^M
index <- spots[1]^M
spots <- index:nrow(object$M)^M
}^M
y <- object$M[spots,j]^M
x <- object$A[spots,j]^M
w <- weights[spots,j]^M
object$M[spots,j] <-
loessFit(y,x,w,span=span,iterations=iterations)$residuals^M
spots <- spots + nspots^M
}^M
}^M
},^M
I am using limma version limma_1.3.13.
Kind regards,
Juerg Straubhaar, PhD
Umass Med
------------------------------
Message: 6
Date: Tue, 20 Jan 2004 14:26:53 -0800
From: "Jim Breaux" <jim.breaux@vialogy.com>
Subject: [BioC] Trouble installing 'makecdfenv'
To: <bioconductor@stat.math.ethz.ch>
Message-ID: <000001c3dfa4$81f3b950$2612d240@ViaChange.com>
Content-Type: text/plain
I am having trouble installing the latest release of 'makecdfenv,' and
I am
hoping someone can help me out. When I tried to install the source
package,
I got the following errors after calling "Rcmd INSTALL
makecdfenv_1.4.1.tar.gz":
--------- Making package makecdfenv ------------
**********************************************
WARNING: this package has a configure script
It probably needs manual configuration
**********************************************
adding build stamp to DESCRIPTION
making DLL ...
making read_cdffile.d from read_cdffile.c
read_cdffile.c:52:19: warning: zlib.h: No such file or directory
gcc -DHAVE_ZLIB=1 -IM:/PROGRA~1/R/rw1081/src/include -Wall -O2 -c
read_cdffile.c -o read_cdffile.o
read_cdffile.c:52:19: zlib.h: No such file or directory
read_cdffile.c: In function `openCELfile':
read_cdffile.c:581: warning: implicit declaration of function `gzopen'
read_cdffile.c:581: warning: assignment makes pointer from integer
without a
cast
read_cdffile.c:586: warning: implicit declaration of function `gzgets'
read_cdffile.c:589: warning: implicit declaration of function
`gzrewind'
read_cdffile.c: In function `openCDFfile':
read_cdffile.c:623: warning: assignment makes pointer from integer
without a
cast
read_cdffile.c: In function `close_affy_file':
read_cdffile.c:663: warning: implicit declaration of function
`gzclose'
read_cdffile.c: In function `readline_affy_file':
read_cdffile.c:679: warning: assignment makes pointer from integer
without a
cast
read_cdffile.c: In function `readQC':
read_cdffile.c:908: warning: unused variable `param_unit'
make[2]: *** [read_cdffile.o] Error 1
make[1]: *** [srcDynlib] Error 2
make: *** [pkg-makecdfenv] Error 2
*** Installation of makecdfenv failed ***
Where is the file 'zlib.h' supposed to come from (it doesn't appear to
be in
'makecdfenv_1.4.1.tar.gz')?
Does anyone have any suggestions that might help me get this to work?
Thanks in advance for your help,
Jim
[[alternative HTML version deleted]]
------------------------------
Message: 7
Date: Tue, 20 Jan 2004 17:47:04 -0500
From: YUK FAI LEUNG <yfleung@mcb.harvard.edu>
Subject: [BioC] Proper pooling design
To: bioconductor@stat.math.ethz.ch
Message-ID: <400DAFE8.5030709@mcb.harvard.edu>
Content-Type: text/plain; charset=us-ascii; format=flowed
Hi there,
I am designing a pilot microarray study on embryoic developmental
mutant
using affy platform. The comparison itself is very simple, the mutant
vs
normal at one time point. Due to various reasons (mostly funding and
limited amount of tissue), I can't start with the "ideal" approach in
which each sample is hybridized to an individual chip.
Since I can easily rear a lot of animals, it seems that pooling is the
only choice for the pilot study. However I am not sure what is the
best
way to allocate the pooled samples to each chip. For example if I want
to do 3 array replicates each for the mutant and control. Is it better
to pool enough samples for 3 arrays and then separate the pooled
sample
in 3 portions for hybridization or just pool different individual
samples for different replicates?
It seems to me that the first way is like getting a group expression
average with accessment of technical variation, while the second
approach can also provide some sort of evalution of biological
variation, abeit an averaged one by the pooling. I suspect the latter
approach is better, and would love to know the suggestions from you.
Thanks!
Fai
--
Yuk Fai Leung
Department of Molecular and Cellular Biology
Harvard University
BL 2079, 16 Divinity Avenue
Cambridge, MA 02138
Tel: 617-495-2599
Fax: 617-496-3321
email: yfleung@mcb.harvard.edu; yfleung@genomicshome.com
URL: http://genomicshome.com
------------------------------
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