[BioC] Single Channel Approach for Agilent Arrays

[Gaj Stan (BIGCAT)]

Dear BioConductor-user,

I've recently tested out Chapter 9 in the Limma user documentation concerning applying single channel approach on Agilent arrays. My experimental setup consists of two different (pooled) food interventions in dye flip using the same control sample (n=4 - For each experiment, 1 array + dye-flip). I assume that my ultimate single-channel design matrix would look like this

	fControl	fFood1		fFood2
(1)	1	0	0
(2)	0	1	0
(3)	0	1	0
(4)	1	0	0
(5)	1	0	0
(6)	0	0	1
(7)	0	0	1
(8)	1	0	0

Where:
Food1_vs_Control.Cy3 (1)
Food1_vs_Control.Cy5 (2)
Control_vs_Food1.Cy3 (3)
Control_vs_Food1.Cy5 (4)
Food2_vs_Control.Cy3 (5)
Food2_vs_Control.Cy5 (6)
Control_vs_Food2.Cy3 (7)
Control_vs_Food2.Cy5 (8)

Question 1: Am I missing any information here? Because if I do this as suggested in the manual, I get a matrix with some extra (and empty) attributes such as assign (1,1,1), contrasts (NULL) and contrasts$f ("contr.treatment"). Are these attributes necessary for further steps? 

Question 2: In my normal two-channel analysis approach I added an extra column in my design file that includes the DyeEffect in the statistical analysis (Ebayes) afterwards. Do I need to include them here as well?

The next part is calculating the intraspotCorrelation, based upon the design file above. When executing, I get the following error: "Missing or Infinite Values found in M or A". This error should indeed occur, since individual spots of bad quality were flagged as NA before normalization, resulting in no M or A value (missing value). Is there a way to omit these spots from the intraspotCorrelation function (By changing them to 0 it might have an effect on the average correlation factor?) or does something else need to be done to solve this?

Any suggestions would be greatly appreciated!

Best wishes,

   Stan


---------------------------------------
Stan Gaj, MSc
PhD Student
Dept. of Human Biology / BiGCaT Bioinformatics / Nutrigenomics Consortium
PO BOX 616
UNS 50 - Box 28
University Maastricht
6200MD Maastricht 
the Netherlands
 
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