[BioC] HTqPCR (plotCtOverview) problem
Michael Muratet
mmuratet at hudsonalpha.org
Mon May 3 16:52:39 CEST 2010
On May 2, 2010, at 3:26 AM, Heidi Dvinge wrote:
>>
>> 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)?
Hi Heidi
I don't understand the purpose and function of 'group'. What would be
the effect, for example, of having four identical sample labels? I
tried using the default and I tried setting it to the sample IDs (all
four) and got the same error.
>
> 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.
I'll send it off directly.
Thanks
Mike
>
> \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
>>
>>
>>
>>
>>
>
>
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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