[BioC] Error Message with RMA normalization in AgiMicroRNA

Henrik Bengtsson hb at stat.berkeley.edu
Tue Jun 8 23:08:22 CEST 2010 

On Tue, Jun 8, 2010 at 10:56 PM, Richard Friedman
<friedman at cancercenter.columbia.edu> wrote:
> Jim and List,
>
>
> On Jun 8, 2010, at 9:15 AM, James W. MacDonald wrote:
>
>> Hi Richard,
>>
>> Richard Friedman wrote:
>>>
>>> Dear Bioconductor List,
>>>        I received an error message when trying to normalize data with
>>>  AgiMicroRMA
>>> > agiMicro.rma=rmaMicroRna(agiMicroraw,
>>> +               normalize=TRUE,
>>> +               background=FALSE)
>>> Error in if (min(dd.aux$Rb) < 0) { :
>>>  missing value where TRUE/FALSE needed
>>> > agiMicro.rma=rmaMicroRna(agiMicroraw,
>>> +               normalize=TRUE,
>>> +               background=TRUE)
>>> Error in if (min(dd.aux$Rb) < 0) { :
>>>  missing value where TRUE/FALSE needed
>>
>> It looks like you have some NA values in agiMicroraw$Rb, so I would check
>> that first. I don't see any code in rmaMicroRna() to gracefully handle
>> missing data, so that is likely the problem.
>
>        I didn't see any NAs in Rb but the maximum value of Rb was  was 65535
> whereas the mean and median were 51 and 168 respectively.
> Does this seem like  a saturation effect?
> If so can mess up the program?
> Also I note negative values for $R.
> Can this mess up the program?
>
>>
>> If there aren't any NA values in agiMicroraw$Rb, then you should try
>> debuggind rmaMicroRna() to see where things are going wrong. Simply start
>> with
>>
>> debug(rmaMicroRna)
>>
>> and then
>>
>> agiMicro.rma <- rmaMicroRna(agiMicroraw, TRUE, FALSE)
>>
>> you will then go into the browser, and can step through the function until
>> you get to
>>
>> ProbeName.rep = unique(dd$genes$ProbeName[duplicated(dd$genes$ProbeName)])
>> dd.aux = dd[1:length(ProbeName.rep), ]
>>
>> at which time you can look for NA values in dd.aux$Rb, and try to track
>> down where they come from. But looking at the code, I don't see any obvious
>> way for them to be produced if not already there.
>
> Pasting from session:
> ######################################################################################
>
> debug: dd.aux = dd[1:length(ProbeName.rep), ]
> Browse[2]> dd.aux$Rb
> Error: object 'dd.aux' not found
> ######################################################################################
>
> For some reason dd.aux was nor created. Can that be the problem?

You have to press ENTER (nothing else) in order to evaluate "dd.aux =
..." when you see:

> debug: dd.aux = dd[1:length(ProbeName.rep), ]
> Browse[2]>

and first then do:

> Browse[2]> dd.aux$Rb

EXAMPLE:

> foo <- function() { a <- 1; b <- 2; }
> debug(foo)
> foo()
debugging in: foo()
debug: {
    a <- 1
    b <- 2
}
Browse[2]>
debug: a <- 1
Browse[2]> a
Error: object 'a' not found
Browse[2]> a
Error: object 'a' not found
Browse[2]> a
Error: object 'a' not found
Browse[2]>          # ENTER <= 'a <- 1' is evaluated here
debug: b <- 2
Browse[2]> a
[1] 1
Browse[2]> b
Error: object 'b' not found
Browse[2]>          # ENTER <= 'b <- 2' is evaluated here
exiting from: foo()
>

/Henrik

>
> Thankls and best wishes,
> Rich
>
>
>>
>> Best,
>>
>> Jim
>>
>>
>>> >
>>>
>>> ################################################################################
>>> My session Inforrmation is:
>>> > 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] AgiMicroRna_1.2.0     preprocessCore_1.10.0 affy_1.26.1
>>>  limma_3.4.0
>>> [5] Biobase_2.8.0
>>> loaded via a namespace (and not attached):
>>> [1] affyio_1.16.0
>>> >
>>>
>>> ################################################################################
>>> My session up to the attempt to normalize is:
>>>
>>> #################################################################################
>>> > targets<-readTargets("Targets.txt",verbose=TRUE)
>>> Target File
>>>                       FileName Treatment GErep
>>> AxCNTx1x1x1     AxCNTx1x1x1.txt     A.CNT     1
>>> AxCNTx2x2x2     AxCNTx2x2x2.txt     A.CNT     1
>>> AxCNTx3x3x3     AxCNTx3x3x3.txt     A.CNT     1
>>> AxCNTx7x4x4     AxCNTx7x4x4.txt     A.CNT     1
>>> BxCCL4x1x1x5   BxCCL4x1x1x5.txt    B.CCL4     2
>>> BxCCL4x2x2x6   BxCCL4x2x2x6.txt    B.CCL4     2
>>> BxCCL4x3x3x7   BxCCL4x3x3x7.txt    B.CCL4     2
>>> BxCCL4x5x4x8   BxCCL4x5x4x8.txt    B.CCL4     2
>>> CxBDLx1x1x9     CxBDLx1x1x9.txt     C.BDL     3
>>> CxBDLx2x2x10   CxBDLx2x2x10.txt     C.BDL     3
>>> CxBDLx3x3x11   CxBDLx3x3x11.txt     C.BDL     3
>>> CxBDLx4x4x12   CxBDLx4x4x12.txt     C.BDL     3
>>> DxaHSCx1x1x13 DxaHSCx1x1x13.txt    D.aHSC     4
>>> DxaHSCx2x2x14 DxaHSCx2x2x14.txt    D.aHSC     4
>>> DxaHSCx3x3x15 DxaHSCx3x3x15.txt    D.aHSC     4
>>> DxaHSCx4x4x16 DxaHSCx4x4x16.txt    D.aHSC     4
>>> > agiMicroraw<-readMicroRnaAFE(targets,verbose=TRUE)
>>> Read AxCNTx1x1x1.txt
>>> Read AxCNTx2x2x2.txt
>>> Read AxCNTx3x3x3.txt
>>> Read AxCNTx7x4x4.txt
>>> Read BxCCL4x1x1x5.txt
>>> Read BxCCL4x2x2x6.txt
>>> Read BxCCL4x3x3x7.txt
>>> Read BxCCL4x5x4x8.txt
>>> Read CxBDLx1x1x9.txt
>>> Read CxBDLx2x2x10.txt
>>> Read CxBDLx3x3x11.txt
>>> Read CxBDLx4x4x12.txt
>>> Read DxaHSCx1x1x13.txt
>>> Read DxaHSCx2x2x14.txt
>>> Read DxaHSCx3x3x15.txt
>>> Read DxaHSCx4x4x16.txt
>>>  RGList:
>>>        dd$R:           'gTotalGeneSignal'
>>>        dd$G:           'gTotalProbeSignal'
>>>        dd$Rb:          'gMeanSignal'
>>>        dd$Gb:          'gProcessedSignal'
>>> > agiMicro.rma=rmaMicroRna(agiMicroraw,
>>> +               normalize=TRUE,
>>> +               background=FALSE)
>>> Error in if (min(dd.aux$Rb) < 0) { :
>>>  missing value where TRUE/FALSE needed
>>>
>>> ################################################################################
>>> Thanks and best wishes,
>>> Rich
>>> ------------------------------------------------------------
>>> Richard A. Friedman, PhD
>>> Associate Research Scientist,
>>> Biomedical Informatics Shared Resource
>>> Herbert Irving Comprehensive Cancer Center (HICCC)
>>> Lecturer,
>>> Department of Biomedical Informatics (DBMI)
>>> Educational Coordinator,
>>> Center for Computational Biology and Bioinformatics (C2B2)/
>>> National Center for Multiscale Analysis of Genomic Networks (MAGNet)
>>> Room 824
>>> Irving Cancer Research Center
>>> Columbia University
>>> 1130 St. Nicholas Ave
>>> New York, NY 10032
>>> (212)851-4765 (voice)
>>> friedman at cancercenter.columbia.edu
>>> http://cancercenter.columbia.edu/~friedman/
>>> In Memoriam,
>>> George Scithers
>>>        [[alternative HTML version deleted]]
>>> _______________________________________________
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>>> https://stat.ethz.ch/mailman/listinfo/bioconductor
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>>
>> --
>> James W. MacDonald, M.S.
>> Biostatistician
>> Douglas Lab
>> University of Michigan
>> Department of Human Genetics
>> 5912 Buhl
>> 1241 E. Catherine St.
>> Ann Arbor MI 48109-5618
>> 734-615-7826
>> **********************************************************
>> Electronic Mail is not secure, may not be read every day, and should not
>> be used for urgent or sensitive issues
>
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