Dear James,



Now I can recognize the meaning of: Error in caseM[hkgs[1], ] : incorrect number of dimensions.

Given your correction, I can now calculate delta delta Cq too.

Also, I have a better understanding of how NormqPCR operates and can generate transformed values.

Thanks for everything.



If I have questions down the road with another, larger dataset, I hope you don't mind if I contact you again.



Respectfully,

Franklin Johnson



________________________________
From: jimrperkins@gmail.com [jimrperkins@gmail.com] on behalf of James Perkins [j.perkins@ucl.ac.uk]
Sent: Wednesday, March 06, 2013 11:53 PM
To: Franklin Johnson [guest]
Cc: Bioconductor@r-project.org; FRANKLIN.JOHNSON@wsu.edu
Subject: Re: [BioC] 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@bioconductor.org<mailto:guest@bioconductor.org>> 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

--
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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@ucl.ac.uk<mailto:j.perkins@ucl.ac.uk>

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