Dear Marcelo, No, there isn't any bug. limma simply expects the Signal Mean to be included as a column in the ImaGene output, which is the default behaviour of ImaGene. You have suppressed the Signal Mean column, so when you use limma you have to specify the columns you want explicitly, which is what you have now done. You took non-default action when you used ImaGene, so you need to take non-default action in limma. Best wishes Gordon > Date: Sun, 20 Sep 2009 02:13:30 -0300 > From: Marcelo Laia <marcelolaia at="" gmail.com=""> > Subject: [BioC] [limma] Possible bug on read.maimages function ? > To: Bioconductor <bioconductor at="" stat.math.ethz.ch=""> > Message-ID: > <a35fc1810909192213x7b18b4b1yccb78cc11949d655 at="" mail.gmail.com=""> > Content-Type: text/plain; charset=ISO-8859-1 > > I think there is a bug on read.maimages limma function. > > I try: > >> targets <- readTargets() >> files <- targets[,c("FileNameCy3","FileNameCy5")] >> RG <- read.maimages(files, source="imagene") > Read header information > Read lamina1Cy3_551.txt > Erro em `[.data.frame`(obj, , columns$f) : undefined columns selected > > After a lot of time, I try: > >> targets <- readTargets() >> files <- targets[,c("FileNameCy3","FileNameCy5")] >> RG <- read.maimages(files, source="imagene",columns=list(f="Signal Median",b="Background Median")) > Read header information > Read lamina1Cy3_551.txt > Read lamina1Cy5_650.txt > Read lamina2Cy3_551.txt > Read lamina2Cy5_650.txt > Read lamina3Cy3_532.txt > Read lamina3Cy5_650.txt > Read lamina5Cy3_532.txt > Read lamina5Cy5_635.txt >> > > Here are more information > >> targets > SlideNumber FileNameCy3 FileNameCy5 Cy3 Cy5 > 1 1 lamina1Cy3_551.txt lamina1Cy5_650.txt Pra Banha > 2 2 lamina2Cy3_551.txt lamina2Cy5_650.txt Pra Banha > 3 3 lamina3Cy3_532.txt lamina3Cy5_650.txt Pra Banha > 4 5 lamina5Cy3_532.txt lamina5Cy5_635.txt Pra Banha >> files > FileNameCy3 FileNameCy5 > 1 lamina1Cy3_551.txt lamina1Cy5_650.txt > 2 lamina2Cy3_551.txt lamina2Cy5_650.txt > 3 lamina3Cy3_532.txt lamina3Cy5_650.txt > 4 lamina5Cy3_532.txt lamina5Cy5_635.txt >> > > Header of my files > > Begin Header > > version 8.0.1 > > Date Thu Jun 11 12:35:08 BRT 2009 > > Image File C:\Users\[snip]\lamina1Cy3_551.tif > > Page 0 > > Page Name > > Inverted false > > Begin Field Dimensions > > Field Metarows Metacols Rows Cols > > A 2 2 32 24 > > B 2 2 32 24 > > End Field Dimensions > > Begin Measurement parameters > > Segmentation Method auto > > Signal Low 0.0 > > Signal High 0.0 > > Background Low 0.0 > > Background High 0.0 > > Background Buffer 2.0 > > Background Width 5.0 > > End Measurement parameters > > Begin Alerts > > Control Type Minimum threshold If tested Percentage allowed If > failed Maximum threshold If tested Percentage allowed If failed CV > threshold If tested If failed > > BLANK 0.0 false 1.0% false 500.0 true 0.1% false 1.0 false false > > POSITIVE 1000.0 true 0.1% false 100000.0 false 1.0% false 1.0 false false > > End Alerts > > Begin Quality Flags > > Begin Flagging Settings > > Empty Spots true Threshold: 0.96 > > Poor Spots true > > Begin Poor Spots Parameters > > Background contamination flag true Threshold: 0.9995 > > Background tested against subgrid data only true > > Signal contamination flag true Threshold: 0.9995 > > Signal contamination test connected to background contamination > threshold true > > Ignored percentage flag true Threshold: 25.2 > > Open perimeter flag true Threshold: 20.0 > > Shape regularity flag true Threshold: 0.6 > > Area To Perimeter Ratio flag false Threshold: 0.7 > > Offset flag true Threshold: 60.0 > > Saturation flag true > > End Poor Spots Parameters > > Negative Spots true > > End Flagging Settings > > Begin Flagged spots > > # of Empty Spots: 2667 > > # of Poor Spots: 288 > > # of Negative Spots: 0 > > # of Manually Flagged Spots: 0 > > End Flagged spots > > > > End Quality Flags > > End Header > > Begin Raw Data > > Field Meta Row Meta Column Row Column Gene ID Annotation 1 Annotation > 2 Flag Signal Median Background Median Signal Mode Background > Mode Signal Area Background Area Signal Total Background Total Signal > Stdev Background Stdev Shape Regularity Ignored Area Spot Area Ignored > Median Area To Perimeter Open > Perimeter XCoord YCoord Diameter Position offset Offset X Offset > Y Expected X Expected Y CM-X CM-Y CM Offset CM Offset-X CM > Offset-Y Min Diam Max Diam Control Failed Control Background > contamination present Signal contamination present Ignored % > failed Open perimeter failed Shape regularity failed Perim- to-area > failed Offset failed Empty spot Negative spot Selected spot Saturated > spot > > >> sessionInfo() > R version 2.9.2 (2009-08-24) > i486-pc-linux-gnu > > locale: > LC_CTYPE=pt_BR.UTF-8;LC_NUMERIC=C;LC_TIME=pt_BR.UTF-8;LC_COLLATE=pt_ BR.UTF-8;LC_MONETARY=C;LC_MESSAGES=pt_BR.UTF-8;LC_PAPER=pt_BR.UTF-8;LC _NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=pt_BR.UTF-8;LC_IDEN TIFICATION=C > > attached base packages: > [1] stats graphics grDevices utils datasets methods base > > other attached packages: > [1] limma_2.18.3 > > loaded via a namespace (and not attached): > [1] tools_2.9.2 >> > > Am I doing any mistake? > > Thank you very much > > -- > Marcelo Luiz de Laia > Universidade do Estado de Santa Catarina > UDESC - www.cav.udesc.br > Lages - SC - Brazil > Linux user number 487797