[BioC] HTqPCR problem with replicates

[Heidi Dvinge]

Hi Marco,

> Hi,
>
> I am using TLDA cards with HTqPCR, the cards have 2 samples for each
> card and the features are in triplicates. I import the card and run
> limma but I realised that the triplicates are kept apart. As you can
> see from the last three lines 18S is presented three times but I would
> like to have a average of the three replicates.
>
In order to use the triplicates you ahve to let limmaCtData() know where
the replicates are. In your case, this would mean setting ndups=3, and
e.g. spacing=1 if the replicates genes are in consecutive order, or
spacing=64 is they're spaced evenly across the 192 wells.

The last example in limmaCtData shows an example of how to reorder the
rows, and use ndups=2. If this doesn't solve your problem, then please let
us know what sort of error or output from limmaCtData you get.

HTH
\Heidi

> qPCRraw= readCtData(files=c("palte_res.txt"), path = NULL, n.features
> = 384, flag = 4, feature = 6, type = 7, position = 3, Ct = 8, header =
> TRUE, SDS = TRUE,  na.value = 40,n.data=2)
>
> ###set the layout (192 for each card)
> sample2a.order <- factor(rep(c("x","Y"), each=192))
> qPCRnew <- changeCtLayout(qPCRraw[,], sample.order = sample2a.order)
> ###change the name
> sampleNames(qPCRnew) <- c("Ctrl_R1","Diuron50_R1","Ctrl_R2","Diuron50_R2")
> #set the group
> groups = factor(c("Ctrl","Diuron50","Ctrl","Diuron50"))
>
> q2.cat=setCategory(qPCRnew, Ct.max = 35,groups = samples,n.category=1)
> q2.cat=filterCtData(qPCRnew,remove.category="Undetermined" ,n.category=1)
> #normalized
> q2.NORM= normalizeCtData(
> q2.cat,norm="deltaCt",deltaCt.genes=c("ABL1-Hs01104728_m1","MRPL19-Hs01040217_m1","HPRT1-Hs03929098_m1"))
>
> ######limma
>
> design <- model.matrix(~0 + groups)
> colnames(design) <- c("Ctrl","Diuron50")
> print(design)
>
> contrasts <- makeContrasts(Ctrl-Diuron50, levels = design)
>
> qDE.limma <- limmaCtData(q2.NORM, design = design, contrasts = contrasts)
>
>  qDE.limma
> $`Ctrl - Diuron50`
>                             genes feature.pos       t.test
> p.value adj.p.value         ddCt         FC   meanTarget
> meanCalibrator
> 10              18S-Hs99999901_s1        <NA> 13.020948738
> 0.0006195627  0.04484078  2.856187444 0.13810262 -12.10495367
> -14.961141111
> 11              18S-Hs99999901_s1        <NA> 11.461949841
> 0.0009341829  0.04484078  2.958459694 0.12865151 -12.17514617
> -15.133605861
> 12              18S-Hs99999901_s1        <NA> 11.851588812
> 0.0008389948  0.04484078  2.681318944 0.15589873 -12.30213467
> -14.98345361
>
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