Here is some advice I got from Gordon Smyth about 2.5 years ago:
My approach to designs like this, at
least as a start, is to try duplicateCorrelation() on each of the
levels separately to get an idea of the strength of the correlation
at each level. Very often, some of the levels are so weak that they
can be ignored.
So, first ignore the dye-swaps and set up your design matrix as if
you have 10 biological replicates of each strain, then use
duplicateCorrelation() on the duplicate spots. Second, ignore the
duplicate spots, use the same design matrix but use
duplicateCorrelation() with the dye-swap pairs as the blocking
variable. If the correlation on the dye-swap pairs is not
substantially negative (~ -0.2??), then you could just ignore it and
use duplicateCorrelation() on the duplicate spots. On the other hand,
if the correlation of the duplicate spots is extremely high, you
could just average the values and then use duplicateCorrelation() on
the dye-swaps. This latter option is my preference; see ?avedups for
an easy way to average the duplicate spots.
At 10:35 AM 11/12/2008, sylvie pinloche wrote:
>I am comparing the transcriptome of two yeast strains, A and B. For
>each strain we have 5 biological replicates.
>The strains are compared to a reference on two-color arrays. For
>each biological replicate, we used two slides, in dye-swap.
>So we have 20 slides :
>slide Cy3 Cy5
>1 A1 ref
>2 ref A1
>3 A2 ref
>4 ref A2
>5 A3 ref
>6 ref A3
>7 A4 ref
>8 ref A4
>9 A5 ref
>10 ref A5
>11 B1 ref
>12 ref B1
>13 B2 ref
>14 ref B2
>15 B3 ref
>16 ref B3
>17 B4 ref
>18 ref B4
>19 B5 ref
>20 ref B5
>In addition, the spots are duplicated on each slide.
>So we have biological replicates, technical replicates (dye-swaps)
>and spot duplication. We are interested in genes differentially
>expressed between both strains.
>I carefully read the Limma User's Guide, but it seems that it's not
>possible to handle duplicated spots and technical replicates
>simultaneously. In addition I'm not sure how to design my contrast
>matrix for this combination of biological and technical replicates
>in a common reference design...
>I would appreciate any help, thank you !
>Sylvie Pinloche - INRA
>Bioconductor mailing list
>Search the archives:
Jenny Drnevich, Ph.D.
Functional Genomics Bioinformatics Specialist
W.M. Keck Center for Comparative and Functional Genomics
Roy J. Carver Biotechnology Center
University of Illinois, Urbana-Champaign
1201 W. Gregory Dr.
Urbana, IL 61801
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