James Perkins | 7 Mar 08:53 2013
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Re: ReadqPCR: [[hkgs,]] incorrect number of dimensions

Hi Franklin,

It looks like the problem is due to you only having 1 replicate for case
and 1 for control, and so it can't work out stats etc. I will work on a fix
for this, in the meantime you can work out delta delta Cq by subracting 1
column from qPCRBatch.dcq from the other (followed by 2^ tranformation).

Cheers, hope that helps,

Jim

On 6 March 2013 19:57, Franklin Johnson [guest] <guest@...>wrote:

>
> Dear James,
>
> Thanks for your time and help with ReadqPCR and NormqPCR.
>
> I have worked a bit more with this NormqPCR;
> Very useful package, especially, for normalizing raw Ct data, determining
> housekeeping genes, etc.
>
> Thus far, I have been successful in working through the vignette.
> Although, I have one last inquiry.
> In order to confirm where the error is coming from, I have deduced my
> input file containing the raw Cq values to 2 conditions, 8 detectors, one
> technical replication.
>
> > qPCRBatch=read.qPCR(qPCRBatch.lv)
> Warning message:
> In read.qPCR(qPCRBatch.lv) :
>   Incompatible phenoData object. Created a new one using sample name data
> derived from raw data.
>
> > exprs(qPCRBatch)
>       caseA control
> actin 20.89   20.91
> lox22 21.36   20.50
> lox23 20.93   21.00
> lox34 20.89   20.80
> lox61 19.50   19.50
> lox62 21.00   25.00
> lox69 19.30   15.00
> lox99 21.10   28.00
>
> > contM=cbind(c(1,0), c(0,1))
> > colnames(contM)=c("interest", "no interest")
> > rownames(contM)=sampleNames(qPCRBatch.lv)
> > contM
>         interest no interest
> caseA          1           0
> control        0           1
>
> > hkgs="actin"
> > ddCq.lv=deltaDeltaCq(qPCRBatch.lv, maxNACase=1, maxNAControl=1,
> hkgs=hkgs, contrastM=contM, case="interest", control="no interest",
> statCalc="arith", hkgCalc="arith")
> Error in caseM[hkgs[1], ] : incorrect number of dimensions
>
> Apart from the vignette, I didn't think I would need to identify hkgs in
> each sample??
>
> What is odd is that I can complete the deltaCq function,
> > qPCRBatch.dcq=deltaCq(qPCRBatch.lv, hkgs=hkgs, calc="arith")
> > exprs(qPCRBatch.dcq)
>       caseA control
> actin  0.00    0.00
> lox22  0.47   -0.41
> lox23  0.04    0.09
> lox34  0.00   -0.11
> lox61 -1.39   -1.41
> lox62  0.11    4.09
> lox69 -1.59   -5.91
> lox99  0.21    7.09
>
> , but not the deltaDeltaCq function.
> This says to me that there is something wrong with my "case" vs. "control"
> comparison in the contrast matrix? However, like the vignette, I have my
> hkgs genes in both samples, as required by the same detectors for all
> samples requisite(which makes sense).
>
> Anyhow, it's been great to work with NormqPCR.
> Hopefully, I can complete it by getting the deltaDeltaCq output.
>
> If you have a chance, can you please take a look at the scripts and
> correct me where I'm wrong.
>
> Respectfully,
> Franklin
>
>
>
>
>  -- output of sessionInfo():
>
> > sessionInfo()
> R version 2.15.1 (2012-06-22)
> Platform: x86_64-pc-mingw32/x64 (64-bit)
>
> locale:
> [1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United
> States.1252    LC_MONETARY=English_United States.1252
> [4] LC_NUMERIC=C                           LC_TIME=English_United
> States.1252
>
> attached base packages:
> [1] stats     graphics  grDevices utils     datasets  methods   base
>
> other attached packages:
> [1] limma_3.14.0       NormqPCR_1.4.0     RColorBrewer_1.0-5
> ReadqPCR_1.4.0     affy_1.36.1        Biobase_2.18.0     BiocGenerics_0.4.0
>
> loaded via a namespace (and not attached):
> [1] affyio_1.26.0         BiocInstaller_1.8.3   preprocessCore_1.20.0
> tools_2.15.1          zlibbioc_1.4.0
>
> --
> Sent via the guest posting facility at bioconductor.org.
>
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-- 
Dr James R Perkins
Institute of Structural and Molecular Biology
Division of Biosciences
University College London
Gower Street
London, WC1E 6BT
UK

email: j.perkins@...

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