[BioC] HTqPCR version and plotCtOverview scaling errors

[Michael Muratet]

On Apr 21, 2010, at 2:46 PM, Heidi Dvinge wrote:

> Hello Mike,
>
> the current HTqPCR version is actually 1.1.4, c.f.
> http://www.ebi.ac.uk/bertone/software.html
>
> That's in the R development version, so it'll be automatically  
> installed
> if you have R-2.11. Technically it should also run with R-2.10 (at  
> least
> it works on my laptop), but since mixing package and R versions is
> generally strongly discouraged, doing so is at your own risk.
>
> I don't recall having made any changes to plotCtOverview though. Can  
> you
> perhaps send the exact command you used?
>
> Regards
> \Heidi

Heidi

Thanks for the reply. I will install the 2.11 version of R. (My 2.10  
install says it's current.)

Here's the command:

  plotCtOverview(x,groups=sampleNames(x),replicates=TRUE,conf.int=TRUE)

It puts the first set of error bars in the correct place, but the  
second set of error bars overlaps white space and the spacing between  
error bars is less than the spacing between the main bars. It's as  
though the SD_ bars that included are throwing off the scale. I can  
send you a PDF of the image if you like.

x is a subset containing the official endogenous control (MammU6) and  
several other small RNAs with multiple copies from a qPCRset object  
that I made by hand (I explain how below) from 7900HT files. I can  
send you a copy of this, too, if you like. I made the object from  
7900HT mouse miRNA files I obtained from the RQ Manager package of SDS  
v2.3:

s2010004660_A <- read.delim("~/Windows_Share/2010004660_A_results.txt",
                             header=TRUE,
                             sep="\t",
                             na.string="Undetermined",
                             skip=36)
s2010004660_B <- read.delim("~/Windows_Share/2010004660_B_results.txt",
                             header=TRUE,
                             sep="\t",
                             na.string="Undetermined",
                             skip=36)

{more lines like the above omitted}

wt_4_x <- c(s2010004660_A$Ct,s2010004660_B$Ct)
mt_4_x <- c(s2010004660_C$Ct,s2010004660_D$Ct)
wt_17_x <- c(s2010004660_E$Ct,s2010004660_F$Ct)
mt_17_x <- c(s2010004660_G$Ct,s2010004660_Ha$Ct)

wt_4_feats <- factor(c(as.character(s2010004660_A 
$Detector),as.character(s2010004660_B$Detector)))
mt_4_feats <- factor(c(as.character(s2010004660_C 
$Detector),as.character(s2010004660_D$Detector)))
wt_17_feats <- factor(c(as.character(s2010004660_E 
$Detector),as.character(s2010004660_F$Detector)))
mt_17_feats <- factor(c(as.character(s2010004660_G 
$Detector),as.character(s2010004660_Ha$Detector)))

qs2010004660 <- new("qPCRset", exprs=exprs)
featureNames(qs2010004660) <- sub("-[[:digit:]]* 
$","",wt_4_feats,perl=TRUE)
featureType(qs2010004660) <- wt_4_tasks
sampleNames(qs2010004660) <- c("WT_4wks", "MT_4wks", "WT_17wks",  
"MT_17wks")

The plates (which I assume are stock items from ABI) are unusual  
relative to what I know from the HTpPCR documentation in that each  
treatment consists of two distinct plates. Plate 1 has unique miRNA  
targets and 4 endogenous control duplicates (MammU6). Plate 2 has a  
couple of hundred miRNA targets in duplicate and the same endogenous  
control. I can send you the layout if I haven't explained it well  
enough.

The plot illustrates the point I'm trying to make that the expression  
of the controls is reasonably constant across the treatments, but I  
can't get the layout right.

Thanks

Mike



>> Greetings
>>
>> I have a problem with the axis scale for the main bar being different
>> than that for the error bars, i.e., the error bars don't line up over
>> the signal bars. I was checking this list for this problem and saw
>> that HTqPCR release version is apparently up to version 1.1.0 whereas
>> the bioconductor site shows 1.0.0. Maybe the new release would fix  
>> the
>> problem, but where would I get it? Updating didn't change the  
>> version.
>> I also am getting main bars of the form SD_myGroups, which I assume  
>> is
>> standard deviation. Can I turn this off?
>>
>> Thanks
>>
>> Mike
>>
>> R version 2.10.1 (2009-12-14)
>> i386-apple-darwin9.8.0
>>
>> locale:
>> [1] en_US.UTF-8/en_US.UTF-8/C/C/en_US.UTF-8/en_US.UTF-8
>>
>> attached base packages:
>> [1] stats     graphics  grDevices utils     datasets  methods   base
>>
>> other attached packages:
>> [1] HTqPCR_1.0.0       limma_3.2.3        RColorBrewer_1.0-2
>> Biobase_2.6.1
>>
>> loaded via a namespace (and not attached):
>> [1] affy_1.24.2          affyio_1.14.0        gdata_2.7.1
>> gplots_2.7.4         gtools_2.6.1         preprocessCore_1.8.0
>> [7] tools_2.10.1
>>
>>
>> Michael Muratet, Ph.D.
>> Senior Scientist
>> HudsonAlpha Institute for Biotechnology
>> mmuratet at hudsonalpha.org
>> (256) 327-0473 (p)
>> (256) 327-0966 (f)
>>
>> Room 4005
>> 601 Genome Way
>> Huntsville, Alabama 35806
>>
>> _______________________________________________
>> Bioconductor mailing list
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>> https://stat.ethz.ch/mailman/listinfo/bioconductor
>> Search the archives:
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>>
>
>

Michael Muratet, Ph.D.
Senior Scientist
HudsonAlpha Institute for Biotechnology
mmuratet at hudsonalpha.org
(256) 327-0473 (p)
(256) 327-0966 (f)

Room 4005
601 Genome Way
Huntsville, Alabama 35806