[BioC] Positive correlation between dye-swap technical replicates
James W. MacDonald
jmacdon at med.umich.edu
Thu Jan 14 17:23:19 CET 2010
Michał Góralski wrote:
> Dear All,
> I have some doubts concerning linear model used in my data analysis. I
> was searching for the answer on the mail list but I didn't find the
> similar case.
> I analyse tobacco roots treated with 2 types of stress: NaCl and CdCl2.
> I have pooled common reference and I have 3 biological replicates of
> treated plants. I also did dye swaps as technical replicate.
> This is my targets file:
> SlideNumber Name FileName Cy3 Cy5
> 13244317 21 13244317.gpr Control NaCl
> 13244318 22 13244318.gpr Control NaCl
> 13244315 23 13244315.gpr Control NaCl
> 13244319 31 13244319.gpr Control CdCl2
> 13244337 32 13244337.gpr Control CdCl2
> 13244316 33 13244316.gpr Control CdCl2
> 13244330 21 13244330.gpr NaCl Control
> 13244329 22 13244329.gpr NaCl Control
> 13244331 23 13244331.gpr NaCl Control
> 13244333 31 13244333.gpr CdCl2 Control
> 13244335 32 13244335.gpr CdCl2 Control
> 13244336 33 13244336.gpr CdCl2 Control
> I did background subtraction with method "normexp" and normalization
> "pronttip loess", without normalization between arrays.
> Now I have the vector indicating biological and technical replicates.
> and create model matrix:
> >design=modelMatrix(targets, ref="Control")
> > design
> CdCl2 NaCl
> [1,] 0 1
> [2,] 0 1
> [3,] 0 1
> [4,] 1 0
> [5,] 1 0
> [6,] 1 0
> [7,] 0 -1
> [8,] 0 -1
> [9,] 0 -1
> [10,] -1 0
> [11,] -1 0
> [12,] -1 0
> I'm interested in such contrasts:
> >cmatrix=makeContrasts(NaCl, CdCl2, NaCl-CdCl2,levels=design)
> > cmatrix
> Levels NaCl CdCl2 NaCl - CdCl2
> CdCl2 0 1 -1
> NaCl 1 0 1
> Object for duplicate correlation with dye-swaps:
> >corfit=duplicateCorrelation(MA, design=design, ndups=1, block=biolrep)
> and the first problem is:
> > corfit$consensus
>  0.3926545
> In limma manual it is written that correlation should be negative for
> dye swaps- why is it positive?- is it a question of wrong model matrix
> or is it something wrong with my samples?
As you note below, this is probably due to a dye-bias. Making some MA
plots should help clarify the problem.
> When I do simple hierarchical clustering of log-ratios:
> The plot divides my arrays in two groups that exactly reflects dye
> swaps. So maybe the model is correct?
> I was thinking also about checking dye effect so I tried with such model:
> > design2=cbind(Dye=1, design)
> > design2
> Dye CdCl2 NaCl
> [1,] 1 0 1
> [2,] 1 0 1
> [3,] 1 0 1
> [4,] 1 1 0
> [5,] 1 1 0
> [6,] 1 1 0
> [7,] 1 0 -1
> [8,] 1 0 -1
> [9,] 1 0 -1
> [10,] 1 -1 0
> [11,] 1 -1 0
> [12,] 1 -1 0
> I'm not sure if I can use such model.
> if I use it:
> > corfit=duplicateCorrelation(MA, design=design2, ndups=1, block=blockrep)
> > corfit$consensus
>  -0.04530506
> The second problem is that each probe on my array is duplicated so in
> the final top table I have each gene doubled- I read it is not possible
> in Limma to analyse both technical duplicates and gene replicas on the
> array. Could you give me any hint how to solve this problem?
Well, that isn't much correlation between the dye-swaps after
controlling for the dye-bias, so you might check the correlation between
the technical replicates. It might be more reasonable to ignore the fact
that the dye-swaps are technical replicates and account for the
intra-slide duplicate correlations instead.
The other possibility is to average the within-slide duplicates and
account for the fact that you have technical replication at the dye-swap
level. But you will have to look at your data to make that call.
> I will be glad for any help in this cases
> Best regards,
> Michal Goralski, PhD student,
> Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan,
> Bioconductor mailing list
> Bioconductor at stat.math.ethz.ch
> Search the archives:
James W. MacDonald, M.S.
University of Michigan
Department of Human Genetics
1241 E. Catherine St.
Ann Arbor MI 48109-5618
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