[BioC] AgiMicroRna - FilterMicroRna question
Neel Aluru
naluru at whoi.edu
Wed Jun 2 14:42:40 CEST 2010
Hi Pedro,
Thank you for the reply. I did try using readMicroRnaAFE function and it gives more error messages. I used Agilent microRNA arrays and scanned them using Axon 4000B scanner. Data was extracted using 9.5. version of Agilent feature extraction software. I am mentioning these just to let you know if any of these make any difference to the data. I have all the columns but for some reason they are not recognized by the program. Here is the session info using readMicroRnaAFE function. It will not calculate CV and also the normalization. That's why I used read.ma images function.
Other than this minor glitch, this package was very easy to understand and use. Thank you very much for writing this package.
Sincerely, Neel
Session info ()
> setwd("/Users/Neel/Desktop/miRNAarray")
> targets.micro=readTargets(infile="targets.txt", verbose=TRUE)
Target File
FileName Treatment GErep Subject
36_DMSO_1 36_DMSO_1.txt 36DMSO 1 1
36_DMSO_2 36_DMSO_2.txt 36DMSO 1 2
36_DMSO_3 36_DMSO_3.txt 36DMSO 1 3
36_TCDD_1 36_TCDD_1.txt 36TCDD 2 1
36_TCDD_2 36_TCDD_2.txt 36TCDD 2 2
36_TCDD_3 36_TCDD_3.txt 36TCDD 2 3
60_DMSO_1 60_DMSO_1.txt 60DMSO 3 1
60_DMSO_2 60_DMSO_2.txt 60DMSO 3 2
60_DMSO_3 60_DMSO_3.txt 60DMSO 3 3
60_TCDD_1 60_TCDD_1.txt 60TCDD 4 1
60_TCDD_2 60_TCDD_2.txt 60TCDD 4 2
60_TCDD_3 60_TCDD_3.txt 60TCDD 4 3
> dd.micro = readMicroRnaAFE(targets.micro,verbose=TRUE)
Read 36_DMSO_1.txt
Read 36_DMSO_2.txt
Read 36_DMSO_3.txt
Read 36_TCDD_1.txt
Read 36_TCDD_2.txt
Read 36_TCDD_3.txt
Read 60_DMSO_1.txt
Read 60_DMSO_2.txt
Read 60_DMSO_3.txt
Read 60_TCDD_1.txt
Read 60_TCDD_2.txt
Read 60_TCDD_3.txt
RGList:
dd$R: 'gTotalGeneSignal'
dd$G: 'gTotalProbeSignal'
dd$Rb: 'gMeanSignal'
dd$Gb: 'gProcessedSignal'
> cvArray(dd.micro, "MeanSignal", targets.micro, verbose=TRUE)
Foreground: MeanSignal
FILTERING BY ControlType FLAG
RAW DATA: 5335
PROBES without CONTROLS: 4620
Error in cvArray(dd.micro, "MeanSignal", targets.micro, verbose = TRUE) :
NOT DUPLICATED ProbeName in chip
> ddTGS.rma = rmaMicroRna(dd.micro, normalize=TRUE, background=FALSE)
Error in if (min(dd.aux$Rb) < 0) { :
missing value where TRUE/FALSE needed
On Jun 2, 2010, at 5:32 AM, Pedro López Romero wrote:
> Hi Neel,
> Try to use readMicroRnaAFE(targets,verbose=TRUE) to load your data into R instead of calling read.maimages() by yourself. This will solve your problem
>
> Cheers
>
> p.-
>
>
> -----Mensaje original-----
> De: Neel Aluru [mailto:naluru at whoi.edu]
> Enviado el: Tuesday, June 01, 2010 7:34 PM
> Para: Martin Morgan
> CC: bioc; Pedro López Romero
> Asunto: Re: [BioC] AgiMicroRna - FilterMicroRna question
>
> Thanks, Martin. I have contacted Pedro today and hopefully he will get a chance to see my mail. In the mean time I will follow your suggestions.
>
> Thanks once again.
>
> Neel
>
> On Jun 1, 2010, at 1:31 PM, Martin Morgan wrote:
>
>> On 06/01/2010 06:43 AM, Neel Aluru wrote:
>>> Hello,
>>>
>>> I have asked this question before and haven't heard from anyone. Sorry for reposting it as I spent lot of time on it and still cannot figure it out. I need to filter the data before statistical analysis so as to remove the genes that are not detected.
>
>>>
>>>> ddPROC = filterMicroRna(ddTGS.rma, dd.micro, control = TRUE,
>>> IsGeneDetected = TRUE, wellaboveNEG = FALSE, limIsGeneDetected = 50,
>>> limNEG = 25, makePLOT = FALSE, targets.micro, verbose = TRUE)
>>> FILTERING PROBES BY FLAGS
>>>
>>>
>>> FILTERING BY ControlType
>>> Error in matrix(ddFILT$other$gIsGeneDetected, nrow = dim(ddFILT)[1],
>>> ncol = dim(ddFILT)[2]) :
>>> attempt to set an attribute on NULL
>>>
>>>
>>> I checked my data files to see if the required column (IsGeneDetected) is present and it is there. But, for some reason it is not detecting and I do not understand the error message I am getting. If anyone can explain the error message to me that would be great. I have posted the session info below.
>>
>> Hi Neel -- I can't help with specifics, but
>>
>>> matrix(NULL)
>> Error in matrix(NULL) : attempt to set an attribute on NULL
>>
>> so the proximate cause of the error message is likely that
>> ddFILT$other$gIsGeneDetected is equal to NULL, e.g., because it doesn't
>> exist. You can investigate this by inspecting the code, e.g.,
>>
>>> options(error=browser())
>>
>> and then re-running your code. See ?browser; when done use
>> options(error=NULL). Before that I'd revisit the help page for this
>> function and double-check that you are providing appropriate arguments.
>>
>> I've added
>>
>>> packageDescription('AgiMicroRna')$Maintainer
>> [1] "Pedro Lopez-Romero <plopez at cnic.es>"
>>
>> to the email, as Pedro in the best position to help you.
>>
>> Martin
>>
>>> Thank you very much,
>>>
>>> Neel
>>>
>>>
>>>
>>>
>>> Session Info
>>>
>>>> library("AgiMicroRna")
>>>> targets.micro=readTargets(infile="targets.txt", verbose=TRUE)
>>>
>>> Target File
>>> FileName Treatment GErep Subject
>>> 36_DMSO_1 36_DMSO_1.txt 36DMSO 1 1
>>> 36_DMSO_2 36_DMSO_2.txt 36DMSO 1 2
>>> 36_DMSO_3 36_DMSO_3.txt 36DMSO 1 3
>>> 36_TCDD_1 36_TCDD_1.txt 36TCDD 2 1
>>> 36_TCDD_2 36_TCDD_2.txt 36TCDD 2 2
>>> 36_TCDD_3 36_TCDD_3.txt 36TCDD 2 3
>>> 60_DMSO_1 60_DMSO_1.txt 60DMSO 3 1
>>> 60_DMSO_2 60_DMSO_2.txt 60DMSO 3 2
>>> 60_DMSO_3 60_DMSO_3.txt 60DMSO 3 3
>>> 60_TCDD_1 60_TCDD_1.txt 60TCDD 4 1
>>> 60_TCDD_2 60_TCDD_2.txt 60TCDD 4 2
>>> 60_TCDD_3 60_TCDD_3.txt 60TCDD 4 3
>>>
>>>> dd.micro=read.maimages(targets.micro$FileName,
>>> columns=list(R="gTotalGeneSignal",G=
>>> "gTotalProbeSignal",Rb="gMeanSignal", Gb="gProcessedSignal"),
>>> annotation=c("ProbeUID","ControlType","ProbeName","GeneName","SystematicName",
>>> "sequence", "accessions","probe_mappings",
>>> "gIsGeneDetected","gIsSaturated","gIsFeatNonUnifOL",
>>> "gIsFeatPopnOL","chr_coord","gBGMedianSignal","gBGUsed"))
>>> Read 36_DMSO_1.txt
>>> Read 36_DMSO_2.txt
>>> Read 36_DMSO_3.txt
>>> Read 36_TCDD_1.txt
>>> Read 36_TCDD_2.txt
>>> Read 36_TCDD_3.txt
>>> Read 60_DMSO_1.txt
>>> Read 60_DMSO_2.txt
>>> Read 60_DMSO_3.txt
>>> Read 60_TCDD_1.txt
>>> Read 60_TCDD_2.txt
>>> Read 60_TCDD_3.txt
>>>> cvArray(dd.micro, "MeanSignal", targets.micro, verbose=TRUE)
>>> Foreground: MeanSignal
>>>
>>> FILTERING BY ControlType FLAG
>>>
>>> RAW DATA: 5335
>>> PROBES without CONTROLS: 4620
>>> ----------------------------------
>>> (Non-CTRL) Unique Probe: 490
>>> (Non-CTRL) Unique Genes: 231
>>> ----------------------------------
>>> DISTRIBUTION OF REPLICATED NonControl Probes
>>> reps
>>> 5 6 7 10
>>> 20 18 36 416
>>> ------------------------------------------------------
>>> Replication at Probe level- MEDIAN CV
>>> 36_DMSO_1 36_DMSO_2 36_DMSO_3 36_TCDD_1 36_TCDD_2 36_TCDD_3 60_DMSO_1
>>> 60_DMSO_2 60_DMSO_3
>>> 0.078 0.081 0.091 0.081 0.077 0.067
>>> 0.076 0.066 0.103
>>> 60_TCDD_1 60_TCDD_2 60_TCDD_3
>>> 0.073 0.086 0.069
>>> ------------------------------------------------------
>>> DISTRIBUTION OF REPLICATED Noncontrol Genes
>>> reps
>>> 20
>>> 231
>>> ------------------------------------------------------
>>>> ddTGS.rma = rmaMicroRna(dd.micro, normalize=TRUE, background=FALSE)
>>> Calculating Expression
>>>> ddPROC = filterMicroRna(ddTGS.rma, dd.micro, control = TRUE,
>>> IsGeneDetected = TRUE, wellaboveNEG = FALSE, limIsGeneDetected = 50,
>>> limNEG = 25, makePLOT = FALSE, targets.micro, verbose = TRUE)
>>> FILTERING PROBES BY FLAGS
>>>
>>>
>>> FILTERING BY ControlType
>>> Error in matrix(ddFILT$other$gIsGeneDetected, nrow = dim(ddFILT)[1],
>>> ncol = dim(ddFILT)[2]) :
>>> attempt to set an attribute on NULL
>>>
>>>> MMM = ddTGS.rma$Rb
>>>> colnames(MMM) = colnames(dd.micro$Rb)
>>>> maintitle='TGS.rma'
>>>> colorfill='blue'
>>>> ddaux=ddTGS.rma
>>>> ddaux$G=MMM
>>>> mvaMicroRna(ddaux, maintitle, verbose=TRUE)
>>>
>>> ------------------------------------------------------
>>> mvaMicroRna info:
>>> FEATURES : 231
>>> POSITIVE CTRL: 12
>>> NEGATIVE CTRL: 7
>>> STRUCTURAL: 3
>>>> rm(ddaux)
>>>> RleMicroRna(MMM,"RLE TGS.rma", colorfill)
>>>> boxplotMicroRna(MMM, maintitle, colorfill)
>>>> plotDensityMicroRna(MMM, maintitle)
>>>> spottypes = readSpotTypes()
>>>> ddTGS.rma$genes$Status = controlStatus(spottypes, ddTGS.rma)
>>> Matching patterns for: ProbeName GeneName
>>> Found 231 gene
>>> Found 1 BLANK
>>> Found 1 Blank
>>> Found 0 blank
>>> Found 6 positive
>>> Found 0 negative
>>> Found 0 flag1
>>> Found 0 flag2
>>> Found 6 flag3
>>> Found 5 flag4
>>> Found 1 flag5
>>> Setting attributes: values
>>>> i = ddTGS.rma$genes$Status == "gene"
>>>> esetPROC = esetMicroRna(ddTGS.rma[i,], targets.micro,
>>> makePLOT=TRUE, verbose = TRUE)
>>> outPUT DATA: esetPROC
>>> Features Samples
>>> 231 12
>>>> design=model.matrix(~-1+treatment)
>>>> print(design)
>>> treatment36DMSO treatment36TCDD treatment60DMSO treatment60TCDD
>>> 1 1 0 0 0
>>> 2 1 0 0 0
>>> 3 1 0 0 0
>>> 4 0 1 0 0
>>> 5 0 1 0 0
>>> 6 0 1 0 0
>>> 7 0 0 1 0
>>> 8 0 0 1 0
>>> 9 0 0 1 0
>>> 10 0 0 0 1
>>> 11 0 0 0 1
>>> 12 0 0 0 1
>>> attr(,"assign")
>>> [1] 1 1 1 1
>>> attr(,"contrasts")
>>> attr(,"contrasts")$treatment
>>> [1] "contr.treatment"
>>>
>>>> fit=lmFit(esetPROC, design)
>>>> cont.matrix = makeContrasts(treatment36TCDDvstreatment36DMSO =
>>> treatment36TCDD-treatment36DMSO, treatment60TCDDvstreatment60DMSO =
>>> treatment60TCDD-treatment60DMSO,treatment60TCDDvstreatment36TCDD =
>>> treatment60TCDD-treatment36TCDD, treatment60DMSOvstreatment36DMSO =
>>> treatment60DMSO-treatment36DMSO, levels=design)
>>>> print(cont.matrix)
>>> Contrasts
>>> Levels treatment36TCDDvstreatment36DMSO
>>> treatment60TCDDvstreatment60DMSO
>>> treatment36DMSO -1
>>> 0
>>> treatment36TCDD 1
>>> 0
>>> treatment60DMSO 0
>>> -1
>>> treatment60TCDD 0
>>> 1
>>> Contrasts
>>> Levels treatment60TCDDvstreatment36TCDD
>>> treatment60DMSOvstreatment36DMSO
>>> treatment36DMSO 0
>>> -1
>>> treatment36TCDD -1
>>> 0
>>> treatment60DMSO 0
>>> 1
>>> treatment60TCDD 1
>>> 0
>>>> fit2 = contrasts.fit(fit,cont.matrix)
>>>> print(head(fit2$coeff))
>>> Contrasts
>>> treatment36TCDDvstreatment36DMSO treatment60TCDDvstreatment60DMSO
>>> dre-let-7a 0.038640984 0.013333873
>>> dre-let-7b 0.074038749 -0.031608286
>>> dre-let-7c 0.026244357 -0.005682488
>>> dre-let-7d 0.067340768 0.055567054
>>> dre-let-7e 0.004569306 0.136348664
>>> dre-let-7f 0.042880109 0.085568058
>>> Contrasts
>>> treatment60TCDDvstreatment36TCDD treatment60DMSOvstreatment36DMSO
>>> dre-let-7a 1.7358343 1.76114142
>>> dre-let-7b 0.1366920 0.24233899
>>> dre-let-7c 0.9920976 1.02402449
>>> dre-let-7d 0.8098432 0.82161694
>>> dre-let-7e 0.1186829 -0.01309647
>>> dre-let-7f 1.1245878 1.08189990
>>>> fit2 = eBayes(fit2)
>>>> fit2 = basicLimma(esetPROC, design, cont.matrix, verbose = TRUE)
>>> DATA
>>> Features Samples
>>> 231 12
>>>
>>>> DE = getDecideTests(fit2, DEmethod = "separate", MTestmethod =
>>> "BH", PVcut = 0.1, verbose = TRUE)
>>>
>>> ------------------------------------------------------
>>> Method for Selecting DEGs: separate
>>> Multiple Testing method: BH - pval 0.1
>>>
>>> treatment36TCDDvstreatment36DMSO treatment60TCDDvstreatment60DMSO
>>> UP 0 5
>>> DOWN 0 1
>>> treatment60TCDDvstreatment36TCDD treatment60DMSOvstreatment36DMSO
>>> UP 56 51
>>> DOWN 80 91
>>> ------------------------------------------------------
>>>> pvalHistogram(fit2, DE, PVcut = 0.1, DEmethod ="separate",
>>> MTestmethod="BH",cont.matrix, verbose= TRUE)
>>>> significantMicroRna(esetPROC, ddTGS.rma, targets.micro, fit2,
>>> cont.matrix, DE, DEmethod = "separate", MTestmethod= "BH", PVcut =
>>> 0.1, Mcut=0, verbose=TRUE)
>>> ------------------------------------------------------
>>> CONTRAST: 1 - treatment36TCDDvstreatment36DMSO
>>>
>>> Error in data.frame(PROBE_ID, as.character(GENE_ID),
>>> as.character(chr_coord), :
>>> arguments imply differing number of rows: 231, 0
>>>
>>>
>>>
>>>
>>> Neel Aluru
>>> Postdoctoral Scholar
>>> Biology Department
>>> Woods Hole Oceanographic Institution
>>> Woods Hole, MA 02543
>>> USA
>>> 508-289-3607
>>>
>>> _______________________________________________
>>> Bioconductor mailing list
>>> Bioconductor at stat.math.ethz.ch
>>> https://stat.ethz.ch/mailman/listinfo/bioconductor
>>> Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor
>>
>>
>> --
>> Martin Morgan
>> Computational Biology / Fred Hutchinson Cancer Research Center
>> 1100 Fairview Ave. N.
>> PO Box 19024 Seattle, WA 98109
>>
>> Location: Arnold Building M1 B861
>> Phone: (206) 667-2793
>>
>
> Neel Aluru
> Postdoctoral Scholar
> Biology Department
> Woods Hole Oceanographic Institution
> Woods Hole, MA 02543
> USA
> 508-289-3607
>
>
>
>
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Neel Aluru
Postdoctoral Scholar
Biology Department
Woods Hole Oceanographic Institution
Woods Hole, MA 02543
USA
508-289-3607
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