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@wur.nl
internet:   http://nutrigene.4t.com
http://scholar.google.com/citations?user=qFHaMnoAAAAJ
http://www.researcherid.com/rid/F-4912-2010


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