[BioC] HTqPCR (plotCtOverview) problem

Tue May 4 13:24:08 CEST 2010

Hi Mike,

congratulations, you hereby have the dubious honour of having  
uncovered the most obscure bug in HTqPCR yet.

[snip]

>>>> 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.

The purpose of the group parameter is to tell plotCtOverview if any  
of the sample should be treated as biological replicates, in addition  
to the technical replicates that might be present on each plate. So  
if you have e.g. 4 samples and say groups=c("A", "B", "C", "D") then  
they're each treated individually, and only replicates features on  
each plate are considered. However, if you say e.g. groups=c("WT",  
"WT", "WT", "mutant"), then the first 3 are treated as biological  
replicates; hence for each gene you get one bar for WT and one for  
mutant.

However, it turns out that plotCtOverview can't handle sampleNames or  
items in "group" containing a capital M... I'll get that fixed  
obviously, but in the meantime here's what you can do:

genes <- c("snoRNA135-4380912", "snoRNA202-4380914",  
"snoRNA429-4386732")
groups <- c("WT_4wks", "mt_4wks", "WT_17wks", "mt_17wks")  # use mt  
instead of MT
plotCtOverview(s2010004660, genes=genes, conf.int=TRUE,  
groups=groups, xlim=c(0,22))

Note that I've adjusted the xlim in this case to make the legend fit  
in and not overlap with the bars. This could also be done by  
adjusting the y scale:

plotCtOverview(s2010004660, genes=genes, conf.int=TRUE,  
groups=groups, ylim=c(0,26))

Sorry for the hassle caused!
\Heidi

>>
>> 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
>
>
>
>