Hi Gordon, Thank you for your help, I am saying that in previous versions of LimmaGUI I have not had a problem loading and analyzing BlueFuse data. I load the data in following LimmaGUI's series of pop up boxes. First it brings up as box asking where the files are, I tell it where the gal file is and then the targets file and finally the spot types... it then asks what type of results files I have, I click on the other box (because there is no BlueFuse option), and then get the box asking what the column headers are for the foreground and background for each of the channels. I type in AMPCH2 for the Cy5 (foreground and background), then I type in AMPCH1 for Cy3 (fore and background) then it goes onto to the pop up box asking if I want to use background correction, I select no, and then a pop up window appears for spot weighting and If I select yes it tells me that I can only use it when I load the data in using the preset formats (in the window where I select other) for GenePix and yes all other file types except BlueFuse. If I continue it then tells me it will continue without spot weighting... so I always select no spot weighting as why see the error... One on the side problem is that if for example I want to use GenePix data, but not read in the mean data column (that last I knew was preset data column for GenePix files), but rather the median data; I need to use the other function in the results file type so I tell LimmaGUI which columns to look at and then select yes in the spot weighting window it still will not allow me to then say which column has flag data so it cannot use spot weight for GenePix data in this instance...... I have been using LimmaGUI since it came out and over the last few years have had no major problems. Through loading many types of data files and comparing them to the ScanArray files I also have I have narrowed down to what is causing the error. The problem is not in using BlueFuse data files, but rather Fused BlueFuse data files... The fusion combines on slide replicates and out puts a different results file (looks the same just only has one data point per oligo control etc). I am thinking the problem is coming form the fact that the gal file has replicates separated and the results files doesn't. I am not sure why this matters for plotting an M box plot, but do realize it might be a problem later on in analysis (although I cannot use the replicate spot function as our replicates are in the same blocks so I always keep the reps separate). Hope that adds further clarification, Liz -----Original Message----- From: Gordon Smyth [mailto:smyth@wehi.edu.au] Sent: Tuesday, August 02, 2005 6:24 PM To: Brooke-Powell, Elizabeth Cc: bioconductor at stat.math.ethz.ch Subject: RE: BlueFuse in limmaGUI [was LimmaGUI Error] At 01:41 AM 3/08/2005, Brooke-Powell, Elizabeth wrote: >Thank you all for you responses. I am sorry I didn't give enough >information. > >I have done what I have always done with BlueFuse, I have told LimmaGUI not >to use background correction or spot quality calls How exactly have you told it not to background correct? The exact sequence of reading in and dialog boxes. > as it cannot with non >GenePix, Spot etc files. Yes it can, with any program that gives background estimates, i.e., anything other than BlueFuse. > When using the other file type function I set the >foreground and background to be the same header (so the Cy5 foreground and >background are AMPCH2 and cy3 is AMPCH1). If I don't use a header it >recognizes it won't load. This has never been a problem in the past as I >tell it to ignore background. Are you saying that the analysis that you are doing works in a previous version of limmaGUI, but not in the version you are using? Gordon