Dear Wei, Yes, your call of the function works with my dataset. :) FYI: initially I also used the values of the slot 'ControlType', defining the negative ctrls being the -1 probes, but then forgot to explicitly define the 'regular' probes being 0. Thanks, Guido -----Original Message----- From: Wei Shi [mailto:shi@wehi.EDU.AU] Sent: Sunday, December 18, 2011 11:47 To: Hooiveld, Guido Cc: bioconductor (bioconductor at stat.math.ethz.ch) Subject: Re: [BioC] limma: bg correction using neqc() w/ Agilent arrays 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]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:7}}