[BioC] OFF-TOPIC: Affy Scaling Factor and Absent/Present Calls

[Adaikalavan Ramasamy]

Dear all,

I have begun working with some affy data and there are some aspects of
it that are confusing to me. The data was analysed using MAS 5.0 suite
and most of the questions here pertain to single arrays (aka Absolute
Analysis?).

The first 3 question is on Scaling Factors (SF) which is defined as 

	SF = TGT / x_96 	; where x_96 is the 2% trimmed mean 


1. Why does MAS 5.0 apply the calculated SF to the middle 96% only and
not 100% of the data ? Since it is often that SF > 1 and signal > 0 by
affy's definition, the top 2% are being penalized. What is the rational
behind this ?

2. I have read that a SF > 3 generally implies problems with the
experiment. Why is this?

3. How is the target intensity, TGT, chosen ? Values of 100, 200, 500
and even 1500 seem to have been chosen. Or do we choose TGT to ensure SF
< 3 for most of the arrays.


The next 2 are related to the Present/Absent calls provided.

4. I have 74 arrays and on average 50% of the data is missing. What is
the normal/acceptable observed percentage missing.

5. I am removing all the signal data with Absent Call. Is this correct
and is there any other use for Absent Call.


Googling and reading the affy manuals (some parts of it anyway) has not
cleared these confusions. Any answers, advices or references from this
list is much appreciated. Thank you for your time.

Regards, Adai.