Hi Sylvie,

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.

HTH,
Jenny

At 10:35 AM 11/12/2008, sylvie pinloche wrote:
>Hello !
>
>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
>
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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

330 ERML
1201 W. Gregory Dr.
Urbana, IL 61801
USA

ph: 217-244-7355
fax: 217-265-5066
e-mail: drnevich@illinois.edu 
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