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Gaj Stan BIGCAT
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@gaj-stan-bigcat-1591
Last seen 10.2 years ago
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
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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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Tel:? +31 (0)43 3882913
Fax: +31 (0)43 3670976
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