[BioC] Double KO and timecourse design / limma

[Gordon K Smyth]

> Date: Mon, 07 Jan 2013 08:29:17 +0100
> From: Textoris Julien <julien.textoris at gmail.com>
> To: bioconductor at r-project.org
> Subject: [BioC] Double KO and timecourse design / limma
>
> Dear all,
>
> First thing, happy new year to all and thanks for all the
> knowledge/advices brought through the list !
>
> I would like to have your advice/comments on the following design.
>
> I have to analyse a microarray dataset performed on MoGene ST 1.0 which
> comprise 22 arrays. I have allmost no replicates (! could not control
> this !) but I thought I could use the timecourse and double KO/single KO
> status to overcome this ?

Not sure what you mean.  You need replicates.  If you don't have them, 
then show us all 22 rows of the targets frame so we can see what else 
might be done.

> The variables are :
>  - Treatment : binary YES or NO
>  - Time : samples at 0h (= UNTREATED), 2h, 6h and 18h
>  - Strain : Wild type (WT), single KO for gene A or gene B, and double KO.
>
> For WT mice, I have 2 UNTREATED (=0h), and a timecourse for Treatment 1
> and 0 (WT_UNT_2h, and WT_TTT_2h). For KO mice, I only have treated
> samples (except time 0h, which is considered untreated).
>
> I have duplicates only for WT.UNTREATED_0h and KOA_0h.
>
> I wrote the following design :
>
> levels(f) =
> c("WT_0h","WT_2h","WT_6h","WT_18h","WT_UNT_2h","WT_UNT_6h","WT_UNT_18h","KOA_0h","KOA_2h",
> ...,"DKO_0h","DKO_2h","DKO_6h","DKO_18h")
>
> and performed the contrast matrix over time like his :
>
> cont.wt = contrast.matrix(
>       "WT_18h-WT_6h",
>       "WT_6h-WT_2h",
>       "WT_2h-WT_0h",
>       levels=design)
>
> idem for KOA, KOB and DKO, and then the comparisons :
>
> cont.koa.wt = contrast.matrix(
>       "(KOA_18h-KOA_6h)-(WT_18h-WT_6h)",
>       etc...
>
>
> How would you handle the strain variable ? As one variable with four
> levels : WT, KOA, KOB, DKO ? Or is it possible to take into account that
> DKO is somehow like KOA+KOB ? Do I have to transform 'Strain' into three
> binary variables : WT (1/0), KOA(1/0) and KOB (1/0) and code DKO as KOA
> = 1 and KOB = 1 ?

You have already incorporated the strain variable.  There is no need to do 
anything else or to make any transformations.

You can test whether DKO is like KOA+KOB by testing the appropriate 
contrast.

> The second question is I don't know how to integrate the WT mice that
> are untreated ?

By forming contrasts of interest.

Best wishes
Gordon

> This experimental design is a bit too complex for me, so any advice
> would be greatly appreciated !
>
> Thanks in advance,
>
> Julien

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