[BioC] HTqPCR (plotCtOverview) problem
Heidi Dvinge
heidi at ebi.ac.uk
Sun May 2 10:26:40 CEST 2010
>
> On Apr 30, 2010, at 3:08 PM, Heidi Dvinge wrote:
>
>>>
>>> On Apr 30, 2010, at 2:42 PM, Heidi Dvinge wrote:
>>>
>>>> Hello Mike,
>>>>
>>>>> Greetings
>>>>>
>>>>> I have R 2.11, BioC 2.6 and HTqPCR 1.2 (see session info at the
>>>>> end).
>>>>>
>>>>> I created a qPCRset object by using rbind() to concatenate four
>>>>> pairs
>>>>> of dissimilar cards for each experimental condition and then
>>>>> cbind()
>>>>> to concatenate the four objects into one:
>>>>>
>>>>> WT_time1.a |
>>>>> WT_time1.b |---> WT_time1
>>>>> MT_time1.a |
>>>>> MT_time1.b |---> MT_time1
>>>>> WT_time2.a | ---> s2010004660
>>>>> WT_time2.b |---> WT_time2
>>>>> MT_time2.a |
>>>>> MT_time2.b |---> MT_time2
>>>>>
>>>>> I set g <- c("MammU6") which is a endogenous control gene.
>>>>>
>>>>> I then tried to plotCtOverview(s2010004660, genes=g,
>>>>> groups=sampleNames(qPCRset), conf.int=TRUE) and got
>>>>>
>>>>>> plotCtOverview(s2010004660, genes=g, conf.int=TRUE)
>>>>> Error in if (del == 0 && to == 0) return(to) :
>>>>> missing value where TRUE/FALSE needed
>>>>>
>>>>> Here's the object info:
>>>>>
>>>>>> s2010004660
>>>>> An object of class "qPCRset"
>>>>> Size: 768 features, 4 samples
>>>>> Feature types: Endogenous Control, Target
>>>>> Feature names: mmu-let-7b-4373168 mmu-let-7c-4373167 mmu-
>>>>> let-7d-4395394 ...
>>>>> Feature classes:
>>>>> Feature categories: OK, Undetermined
>>>>> Sample names: WT_4wks MT_4wks WT_17wks ...
>>>>>
>>>>> Could it be that the Feature classes slot is empty?
>>>>>
>>>> Feature classes shouldn't matter at all here. I can reproduce this
>>>> using
>>>> the data sets included in HTqPCR (qPCRraw and qPCRpros) though. What
>>>> does
>>>> traceback() say? And does the same happen when you use
>>>> conf.int=FALSE?
>>>
>>> Thanks Heidi
>>>
>>> Here's the traceback():
>>>
>>>> plotCtOverview(s2010004660, genes=g, conf.int=TRUE)
>>> Error in if (del == 0 && to == 0) return(to) :
>>> missing value where TRUE/FALSE needed
>>>> traceback()
>>> 3: seq.default(1.5, ncol(SD) * (nrow(SD) + 1), nrow(SD) + 1)
>>> 2: seq(1.5, ncol(SD) * (nrow(SD) + 1), nrow(SD) + 1)
>>> 1: plotCtOverview(s2010004660, genes = g, conf.int = TRUE)
>>>
>>> conf.int = FALSE it makes the plot without complaining. Actually, it
>>> makes the plot in the first case, too, it just doesn't draw error
>>> bars.
>>>
>> Hm, so for some reason it can't calculate a standard deviation for
>> your
>> qPCRset. However it should work even if SD returns NA.
>>
>> Do your have replicate genes in your set? And what happens if you
>> say e.g.
>> groups=c("A", "A", "B", "B")?
> Heidi
>
> I see the same problem.
>
> > plotCtOverview(s2010004660, genes=g,
> groups=c("WT_4wks"),conf.int=TRUE,replicates=TRUE)
> Error in if (del == 0 && to == 0) return(to) :
> missing value where TRUE/FALSE needed
> > plotCtOverview(s2010004660, genes=g,
> groups=c("WT_4wks","MT_4wks"),conf.int=TRUE,replicates=TRUE)
> Error in if (del == 0 && to == 0) return(to) :
> missing value where TRUE/FALSE needed
>
Just for the record, "group" needs a vector the same length as your number
of samples, so in your case it should be c("WT_4wks","MT_4wks",
"MT_17wks", "MT_17wks"). I'll add a warning about that in plotCtOverview,
but I don't think that's the cause of the problem here. What do you get if
you say summary(s2010004660)?
I can't reproduce this behaviour with any of the data I have, but if you
don't mind sending my your qPCRset (off list), then perhaps I can have a
lot at if directly.
\Heidi
> It makes a plot but without error bars.
>
> Yes, there are replicates:
>
> Each card (stock ABI item) has 4 duplicates of the control.
>
> > exprs(s2010004660)[grep("U6",featureNames(s2010004660)),]
> WT_4wks MT_4wks WT_17wks MT_17wks
> MammU6-4395470 17.06939 17.18261 17.31673 17.11643
> MammU6-4395470 17.15968 17.25446 17.31998 17.10729
> MammU6-4395470 17.06865 17.07786 17.22182 17.02346
> MammU6-4395470 17.20133 17.17483 17.11134 16.96466
> MammU6-4395470 17.99183 17.92814 18.57421 18.11891
> MammU6-4395470 17.72530 17.83194 18.60460 17.95789
> MammU6-4395470 17.98547 17.90774 18.75409 18.00277
> MammU6-4395470 17.88600 17.96328 18.40904 18.12783
>
> With stats:
>
> > colMeans(exprs(s2010004660)[grep("U6",featureNames(s2010004660)),])
> WT_4wks MT_4wks WT_17wks MT_17wks
> 17.51096 17.54011 17.91398 17.55241
> > sd(exprs(s2010004660)[grep("U6",featureNames(s2010004660)),])
> WT_4wks MT_4wks WT_17wks MT_17wks
> 0.4230635 0.3975762 0.7266933 0.5388762
>
> Mike
>
>>
>> \Heidi
>>
>>> Thanks again for the package and the help
>>>
>>> Mike
>>>>
>>>> \Heidi
>>>>
>>>>> Here's session info:
>>>>>
>>>>>> sessionInfo()
>>>>> R version 2.11.0 (2010-04-22)
>>>>> 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_1.2.0 limma_3.4.0 RColorBrewer_1.0-2
>>>>> Biobase_2.8.0 MASS_7.3-5
>>>>>
>>>>> loaded via a namespace (and not attached):
>>>>> [1] affy_1.26.0 affyio_1.16.0 gdata_2.7.1
>>>>> gplots_2.7.4 gtools_2.6.1 preprocessCore_1.10.0
>>>>> [7] tools_2.11.0
>>>>>
>>>>> Thanks
>>>>>
>>>>> Mike
>>>>>
>>>>> 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
>>>>>
>>>>>
>>>>>
>>>>>
>>>>>
>>>>
>>>>
>>>
>>> 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
>>>
>>>
>>>
>>>
>>>
>>
>>
>
> 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
>
>
>
>
>
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