[BioC] RE:proper pooling design
Stephen Moore
Stephen.Moore at arragen.com
Wed Jan 21 09:37:31 MET 2004
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 at stat.math.ethz.ch
[mailto:bioconductor-request at stat.math.ethz.ch]
Sent: 20 January 2004 22:49
To: bioconductor at stat.math.ethz.ch
Subject: [SPAM] - Bioconductor Digest, Vol 11, Issue 27 - Email found in
subject
Send Bioconductor mailing list submissions to
bioconductor at stat.math.ethz.ch
To subscribe or unsubscribe via the World Wide Web, visit
https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
or, via email, send a message with subject or body 'help' to
bioconductor-request at stat.math.ethz.ch
You can reach the person managing the list at
bioconductor-owner at stat.math.ethz.ch
When replying, please edit your Subject line so it is more specific
than "Re: Contents of Bioconductor digest..."
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 at fcav.unesp.br>
Subject: Re: [BioC] [maNorm] Normalization a complex experiment...
To: bioconductor at stat.math.ethz.ch
Message-ID: <20040120092034.00002c10 at 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 at 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. Bioquímica 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 at yahoo.com
GS>
--
Marcelo Luiz de Laia, M.Sc.
Dep. de Tecnologia, Lab. Bioquímica 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 at yahoo.com
------------------------------
Message: 2
Date: Tue, 20 Jan 2004 12:38:11 -0000
From: Stephen Henderson <s.henderson at ucl.ac.uk>
Subject: [BioC] limma and makeContrasts
To: "'Bioconductor at stat.math.ethz.ch'"
<Bioconductor at stat.math.ethz.ch>
Message-ID:
<E7CF6BC2744CBE41AE4E87635C7C893C1C668E at 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
**********************************************************************
This email and any files transmitted with it are confidentia...{{dropped}}
------------------------------
Message: 3
Date: Tue, 20 Jan 2004 09:42:34 -0500
From: "Simon Lin" <simon.lin at duke.edu>
Subject: [BioC] Re: Blurriness assesment of scanner TIFF files
To: <bioconductor at stat.math.ethz.ch>
Message-ID: <002201c3df63$a46366c0$71761098 at 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 at med.rug.nl>
Subject: [BioC] Blurriness assesment of scanner TIFF files
To: "Bioconductor" <bioconductor at stat.math.ethz.ch>
Message-ID: <002801c3dea2$59154420$a38f7d81 at 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 at 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 at rdls.nestle.com>
Cc: bioconductor at stat.math.ethz.ch
Message-ID: <6.0.0.22.2.20040120093920.01df5b40 at stat.psu.edu>
Content-Type: text/plain; charset="us-ascii"
An HTML attachment was scrubbed...
URL: https://www.stat.math.ethz.ch/pipermail/bioconductor/attachments/20040120/8622c608/attachment-0001.html
------------------------------
Message: 5
Date: Tue, 20 Jan 2004 17:06:25 -0500
From: "Straubhaar, Juerg" <Juerg.Straubhaar at umassmed.edu>
Subject: [BioC] limma: out of bounds error
To: <bioconductor at stat.math.ethz.ch>
Message-ID:
<1A42F1E1A1E73A4F8C6048789F34A32F2D8A99 at 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 at vialogy.com>
Subject: [BioC] Trouble installing 'makecdfenv'
To: <bioconductor at stat.math.ethz.ch>
Message-ID: <000001c3dfa4$81f3b950$2612d240 at 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 at mcb.harvard.edu>
Subject: [BioC] Proper pooling design
To: bioconductor at stat.math.ethz.ch
Message-ID: <400DAFE8.5030709 at 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 at mcb.harvard.edu; yfleung at genomicshome.com
URL: http://genomicshome.com
------------------------------
_______________________________________________
Bioconductor mailing list
Bioconductor at stat.math.ethz.ch
https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor
End of Bioconductor Digest, Vol 11, Issue 27
********************************************
This e-mail is from ArraGen Ltd
The e-mail and any files transmitted with it are confidential and privileged and intended solely for the use of the individual or entity to whom they are addressed.
Any unauthorised direct or indirect dissemination, distribution or copying of this message and any attachments is strictly prohibited.
If you have received the e-mail in error please notify helpdesk at arragen.com or telephone +44 28 38 363841 and delete the e-mail from your system.
E-mail and other communications sent to this company may be reviewed or read by persons other than the intended recipient.
Viruses : although we have taken steps to ensure that this e-mail and any attachments are free from any virus, you should, in keeping with good practice, ensure that they are actually virus free.
ArraGen Ltd. Registration Number NI 43067
Registered Address : Almac House, Charlestown Road, Craigavon, BT63 5UA Northern Ireland
More information about the Bioconductor
mailing list