[BioC] analysis of reference design with even dye-swap across biological replicates

Jenny Drnevich drnevich at illinois.edu
Mon Jun 20 20:46:02 CEST 2011

Hi Nadia,

If the main goal of your experiment is to compare 
each of the treatments to the control, then DO 
NOT pool the control samples! Even though you do 
not care about individual variation, you cannot 
do an accurate statistical test of the difference 
of the means with out the estimate of the 
variance within the controls. Do a standard 
loop-design and make sure the groups are 
dye-balanced (4 replicates in each dye); you do 
not need to do technical dye-swaps to account for 
the dye effect in the model. This will give 4 
groups * 8 replicates / 2 channels = 16 arrays.

That's my 2-cents,

At 01:31 PM 6/20/2011, Aubin-Horth Nadia wrote:
>Hi everybody,
>I am planning to analyse a microarray experiment (Agilent, 2 colors)
>and I would like to make sure I can include dye effect with the hyb
>design used.
>I have 4 groups: a control group ("wild type") and 3 treatments. We
>are interested by the effect of each treatment on gene expression
>compared to the control. My plan is to maximize the statistical power
>to find differences between the control and each treatment by using a
>reference design and having the control in each hyb. Of course, I
>loose statistical power to find differences between treatments.
>I have 8 biological replicates (fish) per group available.
>I am interested to know if I can correctly take dye-bias into account
>using LIMMA and the following design. I am not interested in
>individual gene expression level, only mean and variance for each
>The 24 hybs are performed using the control group (all 8 individuals
>pooled) as the reference and the 8 individuals from each of the 3
>treatments used in only one hyb (no technical replicate). For each
>treatment, 4 biological replicates would be labelled in cye 3 and 4
>biological replicates would be labelled in cy5 (assigned at random
>within treatment). I would thus get an even design in terms of dye
>labelling for the reference and the treatments, 
>but no dye swap/ technical replicate for a 
>specific fish. The goal is to capture as
>much biological variance here (8 fish instead of 4 fish with dye swap)
>for the 24 hybs we can do.
>The target file would look like this (T1, T2 and T3 are treatments and
>the following number represents a biological replicate)
>HYB     CY3             Cy5
>1               ref             T1.1
>2               ref             T1.2
>3               ref             T1.3
>4               ref             T1.4
>5               T1.5            ref
>6               T1.6            ref
>7               T1.7            ref
>8               T1.8            ref
>9               ref             T2.1
>10              ref             T2.2
>11              ref             T2.3
>12              ref             T2.4
>13              T2.5            ref
>14              T2.6            ref
>15              T2.7            ref
>16              T2.8            ref
>17              ref             T3.1
>18              ref             T3.2
>19              ref             T3.3
>20              ref             T3.4
>21              T3.5            ref
>22              T3.6            ref
>23              T3.7            ref
>24              T3.8            ref
>The comparison of interest is the average difference between the
>control and a given treatment , including dye effects
>I thought I could then use the example as in section 7.3 of limma user
>guide on common reference design but including multiple biological
>replicates and a dye effect (from section 8.2)
>Here the contrast matrix is made for treatment 1, T1
>design <- modelMatrix(targets, ref = "ref")
>design <- cbind(Dye = 1, design)
>fit <- lmFit(MA, design)
>cont.matrix <- 
>makeContrasts((T1.1+T1.2+T1.3+T1.4+T1.5+T1.6+T1.7+T1.8)/ 8, levels = design)
>fit2 <- contrasts.fit(fit, cont.matrix)
>fit2 <- eBayes(fit2)
>topTable(fit2, adjust = "BH")
>Could someone please tell me if
>1) the contrast is appropriate?
>2) it will be possible to estimate the dye effect as presented in the
>manual with my own hybridization design?
>The hybs have not been performed yet but I assume that one can still
>tell if the design is balanced. I could use a loop design as is
>normally used in our lab but as I simply want to know what is the
>effect of each treatment, I though a reference design was appropriate,
>especially with such a large number of biological replicates.
>Thank you!
>Nadia Aubin-Horth
>Assistant professor
>Biology Department
>Institute of Integrative and Systems Biology
>Room 1241, Charles-Eugène-Marchand Building
>1030, Ave. de la Médecine
>Laval University
>Quebec City (QC) G1V 0A6
>Phone: 418.656.3316
>Fax: 418.656.7176
>web page: http://wikiaubinhorth.ibis.ulaval.ca/Main_Page
>Bioconductor mailing list
>Bioconductor at r-project.org
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