[BioC] HTqPCR: Calculate CV between replicates with in a plate

[jeremy wilson]

Thanks for your reply Dr.Heidi,

the input files are from ABI RQ analysis (tab delimited text files). I
can directly read these files using the readCtData function.
I see discussions about not able to read files from RQ analysis output
if the text file is a compilation of more than 1 sample. I have the
same problem. I use the development version of R and the HTqPCR.
Please let me know if I am doing anything wrong.

> dat = readCtData(files, path = path, SDS=TRUE, header=TRUE)
> dat
An object of class "qPCRset"
Size:  384 features, 1 samples
Feature types:           Endogenous Control, Target
Feature names:           ITGA4-Hs00168433_m1 ITGA4-Hs00168433_m1
ITGA4-Hs00168433_m1 ...
Feature classes:
Feature categories:      OK, Undetermined
Sample names:            Test2_multipleplates NA NA ...

The file contains two plates (samples). It should show Size:  384
features, 2 samples

 sessionInfo()
R version 2.11.0 Under development (unstable) (2010-02-10 r51118)
i386-pc-mingw32

locale:
[1] LC_COLLATE=English_United States.1252
[2] LC_CTYPE=English_United States.1252
[3] LC_MONETARY=English_United States.1252
[4] LC_NUMERIC=C
[5] LC_TIME=English_United States.1252

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base

other attached packages:
[1] HTqPCR_1.1.0       limma_3.3.9        RColorBrewer_1.0-2 Biobase_2.7.4

loaded via a namespace (and not attached):
[1] affy_1.25.2          affyio_1.15.2        gdata_2.7.1
[4] gplots_2.7.4         gtools_2.6.1         preprocessCore_1.9.0
[7] tools_2.11.0

Thank you

On Sat, Feb 13, 2010 at 1:23 AM, Heidi Dvinge <heidi at ebi.ac.uk> wrote:
> Hello Jeremy,
>
> there's no default way for calculating within-plate CVs in HTqPCR, since
> it'll depend on the exact layout of the plate. Below is an example of how
> this can be done, using the dataframe "design" to indicate how wells on
> the plate belong together. In your case it might be different of course.
> How is this information stored in the input data files and/or the qPCRset
> object?
>
>> library(HTqPCR)
>> # Use test data from the package as example here, with 6 plates
>> data(qPCRraw)
>> sampleNames(qPCRraw)  <- paste("Plate", 1:6, sep="")
>> # What's the order of genes/rep/samples
>> design        <- data.frame(Sample=paste("S", rep(1:4, each=96), sep="_"),
> +               Gene=paste("Gene", rep(rep(1:32, each=3),4), sep="_"),
> +               Replicate=paste("Rep", rep(1:3, 128), sep="_"))
>> # Calculate the CV
>> sd.gene       <- aggregate(exprs(qPCRraw), by=list(design$Sample,
> design$Gene), sd)
>> mean.gene <- aggregate(exprs(qPCRraw), by=list(design$Sample,
> design$Gene), mean)
>> cv.gene       <- sd.gene[,-c(1:2)]/mean.gene[,-c(1:2)]
>> rownames(cv.gene)     <- paste(sd.gene[,1], sd.gene[,2], sep=":")
>
> HTH
> \Heidi
>
>
>> Dear list,
>>
>> I see that the package considers each plate as different sample and
>> calculates CV  for genes across plates (samples) but not with in a
>> plate.
>> Unfortunately my plate design is different. I have 384 well plates
>> with 3 replicates for 32 genes in first 4 rows of the plate. Likewise
>> other 3 samples in the next 3 sections of 4 rows. So in total I have 4
>> different clinical samples on a single plate.
>> 32genes*3replicates*4samples=384 wells.
>> Now I have gene 1 for sample 1 in first 3 wells of a plate. I want to
>> calculate CV for this gene in 3 wells. Similarly for the remaining 32
>> genes of the sample. Like wise I need to calculate CV for the same
>> genes in 3 more samples in the same plate. I see that the package has
>> no function to calculate CVs for plate in this pattern. My apologies
>> if I am missing any thing. I would really appreciate any suggestions
>> or else I will have to write my own script to analyze my Hi-Throughput
>> data.
>>
>> Thank you
>>
>> _______________________________________________
>> Bioconductor mailing list
>> Bioconductor at stat.math.ethz.ch
>> https://stat.ethz.ch/mailman/listinfo/bioconductor
>> Search the archives:
>> http://news.gmane.org/gmane.science.biology.informatics.conductor
>>
>
>
>