[BioC] RNA degradation (Chris Paulse)
Vawter, Marquis
mvawter at uci.edu
Mon Aug 4 11:03:15 MEST 2003
Hi Chris, we have seen the same phenomenon with affyRNA degradation plots.
There is definitely a smooth trend, until the last point. It is very
reproducible, in fact this position should show the highest intensity
overall, but shows about 50% of what would be expected.
Best, Mark
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Today's Topics:
1. marrayNorm - Getting Data Out (michael watson (IAH-C))
2. RNA degradation (Chris Paulse)
3. Repost: marrayNorm 1.1.3 gets stuck (Rob Dunne)
4. Re: Spelling mistakes and some questions re limma (Gordon Smyth)
5. Re: RMA t-test (Rafael A. Irizarry)
6. Re: Repost: marrayNorm 1.1.3 gets stuck (Gordon Smyth)
----------------------------------------------------------------------
Message: 1
Date: Thu, 31 Jul 2003 11:16:02 +0100
From: "michael watson (IAH-C)" <michael.watson at bbsrc.ac.uk>
Subject: [BioC] marrayNorm - Getting Data Out
To: Bioconductor mailing list <bioconductor at stat.math.ethz.ch>
Message-ID:
<20B7EB075F2D4542AFFAF813E98ACD9301C0099D at cl-exsrv1.irad.bbsrc.ac.uk>
Content-Type: text/plain; charset="iso-8859-1"
Hi
This sounds kind of stupid, but if I have my data in an marrayNorm object,
can anyone give any pointers on how to get it out into, say, a tab-delimited
text file?
Are there any special functions or is it simply write.table() ..??
Thanks
Mick
------------------------------
Message: 2
Date: Thu, 31 Jul 2003 15:18:25 -0700
From: "Chris Paulse" <chrispaulse at hotmail.com>
Subject: [BioC] RNA degradation
To: bioconductor at stat.math.ethz.ch
Message-ID: <BAY8-F114Y0OM3G80cE00005707 at hotmail.com>
Content-Type: text/plain; format=flowed
Hi,
How much faith should I place in the p-values reported by summaryAffyRNAdeg?
The plots of average probe intensity vs probe number (5'<-->3') for some
chips I have show a definite positive trend, but usually the last one or two
data points drive the curve negative. Does this correspond to any known
phenomenon?
Thanks,
Chris Paulse
------------------------------
Message: 3
Date: Fri, 1 Aug 2003 08:24:07 +1000 (EST)
From: Rob Dunne <Rob.Dunne at csiro.au>
Subject: [BioC] Repost: marrayNorm 1.1.3 gets stuck
To: bioconductor at stat.math.ethz.ch
Message-ID: <16169.38663.319920.3044 at pride.nsw.cmis.CSIRO.AU>
Content-Type: text/plain; charset=us-ascii
Hi List,
Please excuse the repost. No one responded to my
previous post -- and it seems to me to be quite
important.
The problem is the new marrayNorm 1.1.3
(installed with bioconductor 1.2) -- which seems to get
stuck in an endless loop
marrayNorm 1.1.3 (installed with bioconductor 1.2)
> unix.time(experiment1.norm<-maNorm(experiment1, norm="loess"))
Timing stopped at: 8874.73 19.65 10289.06 0 0
ie I interrupted the process -
but with marrayNorm 1.1.1 reinstalled
marrayNorm 1.1.1
> unix.time(experiment1.norm<-maNorm(experiment1, norm="loess"))
[1] 803.99 29.73 843.40 0.00 0.00
is there a known problem with this package?
bye
rob
--
Rob Dunne Fax: +61 2 9325 3200 Tel: +61 2 9325 3263
CSIRO Mathematical and Information Sciences +61 2 9325 3100
Locked Bag 17, North Ryde, New South Wales, Australia, 1670
http://matilda.vu.edu.au/~dunne Email: Rob.Dunne at csiro.au
Java has certainly revolutionized marketing and litigation.
------------------------------
Message: 4
Date: Fri, 01 Aug 2003 12:09:54 +1000
From: Gordon Smyth <smyth at wehi.edu.au>
Subject: [BioC] Re: Spelling mistakes and some questions re limma
To: "Dave Waddell" <dwaddell at nutecsciences.com>
Cc: BioC Mailing List <bioconductor at stat.math.ethz.ch>
Message-ID: <5.2.0.9.1.20030801111855.00aed328 at imaphost.wehi.edu.au>
Content-Type: text/plain; charset="us-ascii"; format=flowed
Dear Dave,
At 10:45 PM 31/07/2003, Dave Waddell wrote:
>You have a couple of spelling mistakes in the page:
>
><http://bioinf.wehi.edu.au/limma/library/limma/html/5linearmodels.html>http
://bioinf.wehi.edu.au/limma/library/limma/html/5linearmodels.html
>
>estime and explanded
Thanks for letting me know. These typos have been corrected in later
versions of limma.
>Can you point me to a place that would more fully explain the design
>matrix and contrasts with respect to 2-colour dye experiments?
My best suggestion at this time is:
Yang, Y. H., and Speed, T. P. (2003). Design and analysis of comparative
microarray experiments. In T. P. Speed (ed.), Statistical Analysis of Gene
Expression Microarray Data. Chapman & Hall/CRC Press, pages 35-91.
But basically limma is breaking new ground here so there are no good
references for this stuff apart from the User's Guide itself. I am working
on providing more user friendly interfaces to create design and contrast
matrices and more documentation, but obviously these things take time. In
the meantime, a local statistician would be able to give you some help. Or
you could ask for help on bioconductor about specific designs.
> In some Bioconductor packages, the design matrix appears to be
> applicable to the Cy3/Cy5 experiment as a whole and in others to the
> individual Cy3 and Cy5 experiments.
I am not clear what you mean here. As far as I know, limma is the only
package to have the concept of a design matrix and limma is designed to
analyze the whole experiment at once. Other packages basically assume you
are making only one comparison usually with replicate arrays.
> It is very confusing. In addition, the meaning of a contrasts matrix and
> how to put one together is not very clear. Both of these values, if
> applied incorrectly, would appear to me (as a non-statistician assigned
> to put together a package) to completely change the results.
Yes, this is true.
> Finally, can you tell me how limma handles control spots?
The only explicit handling of control spots in limma is in the plotMA
function. I assume that you will leave the control spots in during the
normalization (perhaps using weights to downweight ratio controls spots or
to upweight MSP titration spots) and you will remove them before doing
inference about differential expression. There are subsetting commands to
make removing control spots easy.
>Thanks for a great package, Dave.
Thanks for your comments.
Gordon
------------------------------
Message: 5
Date: Thu, 31 Jul 2003 22:31:13 -0400 (EDT)
From: "Rafael A. Irizarry" <ririzarr at jhsph.edu>
Subject: Re: [BioC] RMA t-test
To: James MacDonald <jmacdon at med.umich.edu>
Cc: bioconductor at stat.math.ethz.ch, dgrigor1 at jhmi.edu
Message-ID:
<Pine.GSO.4.10.10307312222420.8322-100000 at athena.biostat.jhsph.edu>
Content-Type: TEXT/PLAIN; charset=US-ASCII
hi! fyi, all the ROC curves in the NAR paper comparing expression measures
was done on 1-1 comps (i.e. 2 chips). rma seems to perform weel
according to these curves. notice that if you only have one
chip median polish turns into a median which in my opinion is not a
terrible thing to use.
as for t-tests with two arrays all you could use for estimating
variability (denominator in the t-test) are the residuals from the median
polish fit. these have (at least) two problemsB
1) as james points out they have no information about biological
variation
2) its not clear what the statistical properites of these residuals are.
the se estimate one gets with expresso-rma are given only as an ad-hoc
estimate of uncertainty of the expression estimates due to technology.
hope this helps,
rafael
On Mon, 28 Jul 2003, James MacDonald wrote:
> I would be cautious about using RMA with only two chips. You will be
> estimating the probe-specific intensity with only two observations, so
> it is doubtful that the estimate will be very accurate. I recall reading
> somewhere that a good minimum number of chips is around 5-6 for RMA.
>
> As for a t-test with only one observation per group, this is not
> possible. How are you going to estimate the variance for each group?
> Without replication all you can do is ratios, and you are then stuck
> with the assumption that large ratios equal significant differences.
>
> Jim
>
>
>
>
> James W. MacDonald
> Affymetrix and cDNA Microarray Core
> University of Michigan Cancer Center
> 1500 E. Medical Center Drive
> 7410 CCGC
> Ann Arbor MI 48109
> 734-647-5623
>
> >>> DMITRY GRIGORYEV <dgrigor1 at jhmi.edu> 07/28/03 12:08PM >>>
> Hi everyone.
>
> One quick question.
> When I run RMA on just two chips, how could I conduct pairwise t-test
> for each probe set between these chips?
>
> Thank you
>
> Dima
>
> _______________________________________________
> Bioconductor mailing list
> Bioconductor at stat.math.ethz.ch
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>
------------------------------
Message: 6
Date: Fri, 01 Aug 2003 15:30:19 +1000
From: Gordon Smyth <smyth at wehi.edu.au>
Subject: Re: [BioC] Repost: marrayNorm 1.1.3 gets stuck
To: Rob.Dunne at csiro.au
Cc: BioC Mailing List <bioconductor at stat.math.ethz.ch>
Message-ID: <5.2.1.1.1.20030801151709.00b1ee48 at imaphost.wehi.edu.au>
Content-Type: text/plain; charset="us-ascii"; format=flowed
>Hi List,
>
>Please excuse the repost. No one responded to my
>previous post -- and it seems to me to be quite
>important.
>
>The problem is the new marrayNorm 1.1.3
>(installed with bioconductor 1.2) -- which seems to get
>stuck in an endless loop
It isn't in an endless loop, it's just very slow.
The problem is the loess function. If you use family="symmetric" and
iterations=4 to get a robust loess curve rather than just least squares,
then the function is quite slow with lots of data and you get heaps of
warnings associated with memory limits and the use of a k-d tree. If you
use surface="direct" to avoid the k-d tree and stop the warnings, then the
function is very slow indeed with lots of points. You see the result below
when you try to run it on 30,000 data points.
Versions of marrayNorm prior to 1.1.3 used least squares for the loess
curves - much quicker but not ideal as a normalization tool.
I have used some tricks to avoid this sort of speed degradation in the
limma package. I believe that Jean is in the process of implementing to
same sort of thing in the marrayNorm package.
Regards
Gordon
>marrayNorm 1.1.3 (installed with bioconductor 1.2)
> > unix.time(experiment1.norm<-maNorm(experiment1, norm="loess"))
>Timing stopped at: 8874.73 19.65 10289.06 0 0
>
>ie I interrupted the process -
>but with marrayNorm 1.1.1 reinstalled
>
> marrayNorm 1.1.1
> > unix.time(experiment1.norm<-maNorm(experiment1, norm="loess"))
>[1] 803.99 29.73 843.40 0.00 0.00
>
>
>
>is there a known problem with this package?
>
> bye
> rob
>
>--
>Rob Dunne Fax: +61 2 9325 3200 Tel: +61 2 9325 3263
>CSIRO Mathematical and Information Sciences +61 2 9325 3100
>Locked Bag 17, North Ryde, New South Wales, Australia, 1670
><http://matilda.vu.edu.au/~dunne>http://matilda.vu.edu.au/~dunne Email:
><https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor>Rob.Dunne at
>csiro.au
------------------------------
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