[BioC] HTqPCR (plotCtOverview) problem

[Michael Muratet]

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

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