[BioC] limma: bg correction using neqc() w/ Agilent arrays
Wei Shi
shi at wehi.EDU.AU
Sun Dec 18 11:46:31 CET 2011
Dear Guido,
It seems you did not use the 'status' and 'negctrl' parameters correctly when you called neqc function. Try this:
bg.corr <- neqc(e.raw, status=e.raw$genes$ControlType, negctrl=-1, regular=0)
For Agilent one-color arrays, regular probes, negative control probes and positive control probes have control type values of 0, -1 and 1, respectively.
Let me if this does not work.
Cheers,
Wei
On Dec 17, 2011, at 3:36 AM, Hooiveld, Guido wrote:
> Hi,
> I would like to use limma's neqc function for background correction of a set of single colour Agilent arrays obtained from GEO (GSE27784). However, I am not able to get it to work, likely due to a simple issue which I am not able to tackle. Therefore, any pointers to get it running would be appreciated.
>
> Regards,
> Guido
>
>> library("limma")
>>
>> targets <- readTargets("targets_GSE27784.txt", row.names="Name")
>> e.raw <- read.maimages(targets$FileName, source="agilent.median", green.only=TRUE)
> Read GSM686624_251486829200_S01_GE1_105_Jan09_1_1.txt
> Read GSM686625_251486829201_S01_GE1_105_Jan09_1_2.txt
> Read GSM686626_251486829328_S01_GE1_105_Jan09_1_3.txt
> Read GSM686627_251486829200_S01_GE1_105_Jan09_1_2.txt
> Read GSM686628_251486829200_S01_GE1_105_Jan09_1_4.txt
> Read GSM686629_251486829201_S01_GE1_105_Jan09_1_4.txt
> Read GSM686630_251486829328_S01_GE1_105_Jan09_1_4.txt
> Read GSM686631_251486829328_S01_GE1_105_Jan09_1_1.txt
> Read GSM686632_251486829328_S01_GE1_105_Jan09_1_2.txt
> Read GSM686633_251486829200_S01_GE1_105_Jan09_1_3.txt
> Read GSM686634_251486829201_S01_GE1_105_Jan09_1_3.txt
> Read GSM686635_251486829201_S01_GE1_105_Jan09_1_1.txt
>>
>> # Specify neg cons because they are different than what limma expects
>> bg.corr <- neqc(e.raw, status=e.raw$genes$SystematicName, negctrl="NegativeControl")
> Error in if (alpha <= 0) stop("alpha must be positive") :
> missing value where TRUE/FALSE needed
>>
>> # confirm Negative Controls are indeed present in input file
>> e.raw$genes$SystematicName[550:560]
> [1] "NM_001029937" "NM_029372" "NM_178076" "NegativeControl" "NM_032418" "XM_991644"
> [7] "AK036031" "AF012177" "NM_178257" "NM_001038845" "NM_019764"
>>
>> e.raw
> An object of class "EListRaw"
> $E
> GSM686624_251486829200_S01_GE1_105_Jan09_1_1 GSM686625_251486829201_S01_GE1_105_Jan09_1_2 GSM686626_251486829328_S01_GE1_105_Jan09_1_3 GSM686627_251486829200_S01_GE1_105_Jan09_1_2
> [1,] 18713.5 14800 20646.4 19689
> [2,] 27.0 24 25.0 26
> [3,] 24.5 23 22.0 25
> [4,] 26.0 24 23.0 24
> [5,] 26.0 23 23.0 24
> <<SNIP>>
> 45013 more rows ...
>
> $Eb
> GSM686624_251486829200_S01_GE1_105_Jan09_1_1 GSM686625_251486829201_S01_GE1_105_Jan09_1_2 GSM686626_251486829328_S01_GE1_105_Jan09_1_3 GSM686627_251486829200_S01_GE1_105_Jan09_1_2
> [1,] 19 16 17.5 20
> [2,] 20 17 18.0 20
> [3,] 21 17 19.0 20
> [4,] 21 17 18.0 21
> [5,] 20 17 17.0 21
> <<SNIP>>
> 45013 more rows ...
>
> $targets
> [1] "GSM686624_251486829200_S01_GE1_105_Jan09_1_1.txt" "GSM686625_251486829201_S01_GE1_105_Jan09_1_2.txt" "GSM686626_251486829328_S01_GE1_105_Jan09_1_3.txt" "GSM686627_251486829200_S01_GE1_105_Jan09_1_2.txt"
> [5] "GSM686628_251486829200_S01_GE1_105_Jan09_1_4.txt"
> 7 more rows ...
>
> $genes
> Row Col ControlType ProbeName SystematicName
> 1 1 1 1 GE_BrightCorner GE_BrightCorner
> 2 1 2 1 DarkCorner DarkCorner
> 3 1 3 1 DarkCorner DarkCorner
> 4 1 4 1 DarkCorner DarkCorner
> 5 1 5 1 DarkCorner DarkCorner
> 45013 more rows ...
>
> $source
> [1] "agilent.median"
>
>>
>> sessionInfo()
> R version 2.14.0 (2011-10-31)
> Platform: i386-pc-mingw32/i386 (32-bit)
>
> locale:
> [1] LC_COLLATE=English_United States.1252
> [2] LC_CTYPE=English_United States.1252
> [3] LC_MONETARY=English_United States.1252
> [4] LC_NUMERIC=C
> [5] LC_TIME=English_United States.1252
>
> attached base packages:
> [1] stats graphics grDevices utils datasets methods base
>
> other attached packages:
> [1] limma_3.10.0
>>
>
> ---------------------------------------------------------
> Guido Hooiveld, PhD
> Nutrition, Metabolism & Genomics Group
> Division of Human Nutrition
> Wageningen University
> Biotechnion, Bomenweg 2
> NL-6703 HD Wageningen
> the Netherlands
> tel: (+)31 317 485788
> fax: (+)31 317 483342
> email: guido.hooiveld at wur.nl
> internet: http://nutrigene.4t.com
> http://scholar.google.com/citations?user=qFHaMnoAAAAJ
> http://www.researcherid.com/rid/F-4912-2010
>
>
> [[alternative HTML version deleted]]
>
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