[BioC] M vs A plot

[michael watson (IAH-C)]

Richard

The nature of any normalisation means that we will always have outliers - those spots that deviate from all the rest.  There could be two reasons - that spot represents a differentially expressed gene or the spot is unreliable and comes from a "bad" spot.

I'd take the common sense approach to these outliers:

i) Check any replicate spots - if all replicate spots are outliers then you have evidence that it's a differentially expressed gene.  However, if the replicates disagree, this is evidence that the outlier comes from an unreliable / bad measurement

ii) Go take a look at the spot on the original image.  Does it look "good"?

You are likely always to find outliers after normalisation.  This is, after all, what we are looking for, isn't it?  The key is to be able to say, when you see an outlier, if that spot is of reliable quality or not.

Thanks
Mick

-----Original Message-----
From: Richard Friedman [mailto:friedman at cancercenter.columbia.edu]
Sent: 29 January 2004 22:26
To: 'Bioconductor Mail List'
Cc: IRA A TABAS
Subject: [BioC] M vs A plot


Dear Bioconductors,

	I have normalized a series of arrays using print-tip normalization.
Where as the systematic error in the unnormalized data was pronounced,
The systematic error on the normalized array was reduced greatly.
The M vs. A curve was flat for most of the 48 print-tips. However for a 
few
printips, for A>12 M deviates from close to zero. in one case, M rises 
as high
as M=1/2. at A=15. This only involves a small fraction of the spots (It 
is hard to
estimate what proportion).

	Does this sound serious?

	If so, what should I do about it?

	Is anyone willing to look at the JPEg file (I did not attach it 
because I don't
know if I am allowed to do so).

Thanks and best wishes,
Rich
------------------------------------------------------------
Richard A. Friedman, PhD
Associate Research Scientist
Herbert Irving Comprehensive Cancer Center
Oncoinformatics Core
Lecturer
Department of Biomedical Informatics
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Columbia University Medical Center
630 W. 168th St.
New York, NY 10032
(212)305-6901 (5-6901) (voice)
friedman at cancercenter.columbia.edu
http://cancercenter.columbia.edu/~friedman/

"Spring, Summer, and Winter.
Then Fall came along,
and that's the end of our song,
and the pigeons never hibernate at all".
-Rose Friedman, age 7
(These are the correct lyrics and supersede
the version previously at the end of my sig)

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