[BioC] HTqPCR problem with creation of qPCRset

[James W. MacDonald]

Hi Elisabeth,

On 2/19/2013 10:25 AM, Elisabeth Gilis wrote:
> Hi,
>
> I'm a PhD student from the University of Ghent, and I'm just starting 
> to learn how to work with R.
> The initial aim is to analyse qPCR data with the package HTqPCR so I 
> read quite a few manuals regarding the program R and HTqPCR.
> My problem is situated right at the beginning of the analysis, namely 
> loading my data.
> I think I got it correctly that I first have to make a so-called 
> "qPCRset" of my data?
> So just to begin easily, I made an Excel file with just 3 columns:
> Sample    Name      Cp
> A1        Actin     17
> A2        Actin     18
> A3        Actin     16
>
> And saved it as a text file "test.qPCR.txt" to be able to import it.
> For this I downloaded "RStudio" and in this program I imported my 
> dataset "test.qPCR.txt.
> (This is what is printed on the command line (not by me, but by the 
> program RStudio):
>> test.qPCR <- read.delim("~/test.qPCR.txt", dec=",")

It's not such a bad idea to make some fake data to practice with. 
However, you have to first ensure that the data conform to what the 
program expects. If you look at the help page for readCtData(), you will 
see that the input file format can be one of many different types. A 
close reading will indicate that the format you have chosen for your 
fake data isn't one of them.

There are often example scripts that are part of a help page that you 
can run, and that holds true for this function. So if you type

example("readCtData")

at the R prompt, you will run the example data and get a qPCRset that 
you can then play around with. You might also look at the vignette for 
this package:

http://bioconductor.org/packages/2.11/bioc/vignettes/HTqPCR/inst/doc/HTqPCR.pdf

which looks pretty helpful to me.

Best,

Jim


>
> This was no problem at all, I actually get a nice data frame (when I 
> type the command >class(test.qPCR)
> I get [1]"data.frame").
> Then I loaded the package HTqPCR and at this point I'm stuck.
> I want to use my dataset that I imported to make a qPCRset, but I have 
> no clue how to do it.
> I tried already some some commands, but they all give me errors, for 
> example:
>
>> raw<-readCtData("test.qPCR.txt")
> Error in `[.data.frame`(sample, , column.info[["Ct"]]) :
>   undefined columns selected
> In addition: Warning message:
> In .readCtPlain(readfile = readfile, header = header, n.features = 
> n.features,  :
>   384 gene names (rows) expected, got 4
>
>
>> raw<-readCtData(test.qPCR)
> Error in read.table(file = file, header = header, sep = sep, quote = 
> quote,  :
>   'file' must be a character string or connection
>
>
>> raw<-readCtData(files:"test.qPCR",path=path,format="plain")
> Error in files:"test.qPCR" : NA/NaN argument
> In addition: Warning messages:
> 1: In files:"test.qPCR" :
>   numerical expression has 2 elements: only the first used
> 2: In readCtData(files:"test.qPCR", path = path, format = "plain") :
>   NAs introduced by coercion
>
> So I'm really stuck here, if somebody would help me to get started, I 
> would be really grateful!
> Kind regards,
> Elisabeth Gilis, PhD student
> Department of Rheumatology
> Ghent University
>
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-- 
James W. MacDonald, M.S.
Biostatistician
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099