From the headers below, it looks like it is a Genepix file. In Genepix you can choose what the headers will say, regarding the wavelength used. You can choose to write F532, or F546... etc. Alternatively, simply manually change that value to that expected by the analysis software (532 for Cy3 and 635 for Cy5). Alexa 546 and Alexa 555 are two common dyes used as substitutes for Cy3, and despite the different names, they really have very similar spectrum to Cy3... so I don't think you need to worry about your lasers, just change the wavelength numbers on the header to 635 for Cy5 and 532 for Cy3 to ensure the files are accepted. Jose Quoting Kasper Daniel Hansen <khansen at="" stat.berkeley.edu="">: > Ok Hua, I have been pointed to the solution. > > You original post has something like > > <snip> > >>>> "Wavelengths=650 546" > > <snip> > >>>> Block Column Row Name ID X Y Dia. F650 Median >>>> F650 Mean F650 SD B650 Median B650 Mean B650 SD % > B650 1SD >>>> % > B650 2SD F650 % Sat. F546 Median F546 Mean F546 SD B546 >>>> Median B546 Mean B546 SD % > B546 1SD % > B546 2SD F546 > > Notice the wavelengths and the column headers (B650, F645) > > You try to read this using > >>>>>> RG <- read.maimages(files, columns=list(R="F532 Mean", G="F635 >>>>>> Mean", Rb="B532 Median", Gb="B635 Median"), wt.fun=wtflags(0.1)) > > That actually works on the file you sent me, because that file has > > "Wavelengths=635 532" > > <snip> > > "Block" "Column" "Row" "Name" "ID" "X" "Y" "Dia." "F635 Median" "F635 > Mean" "F635 SD" "B635 Median" "B635 Mean" "B635 SD" "% > B635 1SD" "% > > B635 2SD" "F635 % Sat." "F532 Median" "F532 Mean" "F532 SD" "B532 > Median" "B532 Mean" "B532 SD" "% > B532 1SD" "% > B532 2SD" "F532 % > Sat." "Ratio of Medians (532/635)" "Ratio of Means (532/635)" "Median > of Ratios (532/635)" "Mean of Ratios (532/635)" "Ratios SD > (532/635)" "Rgn Ratio (532/635)" "Rgn R? (532/635)" "F Pixels" "B > Pixels" "Sum of Medians" "Sum of Means" "Log Ratio (532/635)" "F635 > Median - B635" "F532 Median - B532" "F635 Mean - B635" "F532 Mean - > B532" "F635 Total Intensity" "F532 Total Intensity" "SNR 635" "SNR > 532" "Flags" "Normalize" > > In this file the wavelengths and the column headers (B635, F532) are > different from before (B650, F645). > > My guess is that you (or however did the scanning) have scanned the > arrays with different wavelengths. At least you have different column > headers in the different files. > > I am not an expert on scanning technology, but my guess is that this > will be really hard to fix. If you want to try anyway (in the hope > that normalization will take care of the wavelength issue), you need > to read the files into two different objects in limma and then > subsequently merge them. > > Since this is a user error, the only reason I can imagine for the > code to run on windows and not on linux is that you have two > (different) set of files.... > > /Kasper > > > On Dec 6, 2005, at 2:49 PM, Kasper Daniel Hansen wrote: > >> It parses fine on the linux system I have access to (which is a bit >> different, it is 64 bit, uses limma 2.4.2 and does not run the >> standard release version of R). >> >> I think I may be wrong about the locale stuff. But you can do a bit >> of debuging >> >> 1) check the files argument: is it really the list of files you want >> (no "wrong" files?) >> 2) you migth want to specify the sources argument to the >> read.maimages function >> 3) check that >> R> for(fileName in files) readLines(fileName, n = 100) >> actually runs (this checks thar R can read the header of each file in >> the files vector you defined in your first email. >> 4) try to debug the function to see what is wrong, ie. try >> R> debug(read.maimages) >> now you can step through the function one line at a time. Try to >> figure out what file/ line it bangs out on. >> >> Beyond that I am not sure I can be of much help as I am unable to >> reproduce the problem and I am fairly unfamiliar with read.maimages. >> >> But try step 1-4. >> >> Kasper >> >> On Dec 6, 2005, at 1:29 PM, Hua Weng wrote: >> >>> Dear Kasper: >>> >>> I attaches the file header fro you to review. I highly appreciate >>> your help. >>> >>> Hua >>> >>> -----Original Message----- >>> From: Kasper Daniel Hansen [mailto:khansen at stat.Berkeley.EDU] >>> Sent: Tuesday, December 06, 2005 2:53 PM >>> To: Hua Weng >>> Cc: bioconductor at stat.math.ethz.ch >>> Subject: Re: [BioC] Error message from R2.2.0 on Linux >>> >>> Unfortunately, the mail system has scrambled the specifics of your >>> message. Try doing something like this (on Linux): >>> >>> # head -100 FILENAME > file.header >>> # gzip file.header >>> >>> and then mail me the header. >>> >>> A big change recently have been how R handles character sets, so I am >>> not surprised that this is an issue. I am not sure I can help you in >>> details (you might want to try R-help), but I can help you isolate >>> the problem. Your LANG seems standard. >>> >>> Kasper >>> >>> >>> On Dec 6, 2005, at 12:16 PM, Hua Weng wrote: >>> >>>> >>>> >>>> Dear Kasper: >>>> >>>> Thank you very much for your response. >>>> >>>> When I took a look at #env, my LANG=en_US.UTF-8 >>>> >>>> Actually, I can read these files on the same machine in R1.8.0 >>>> environment. >>>> But in R2.2.0, I got the following locale error. >>>> >>>> The following is the header part of my gpr file: >>>> >>>> ATF 1 >>>> 27 82 >>>> Type=GenePix Results 2 >>>> DateTime=2005/10/01 20:10:53 >>>> Settings=D:\USERS\DeSilva\asitha\GAL Files\good >>>> gals_3Plates_3X2reps\Hyb4_brahma_6hours_01oct05.gps >>>> GalFile=D:\USERS\DeSilva\asitha\GAL Files\good >>>> gals_3Plates_3X2reps >>>> \3_plates_2x3_reps_4x4_pins_printed083005_Modnew7.gal >>>> >>>> PixelSize=10 >>>> "Wavelengths=650 546" >>>> "ImageFiles=D:\USERS\DeSilva\asitha\TIME POINT HYBS\BRAHMA\FIBRO Vs >>>> 6HRS\TIF >>>> SCAN1\Alexa 647_P60_L78_Brahma_Hyb4_6Hrs_13300853_01oct05.tif 0 >>>> D:\USERS\DeSilva\asitha\TIME POINT HYBS\BRAHMA\FIBRO Vs 6HRS\TIF >>>> SCAN1\Alexa >>>> 546_P66_L79_Brahma_Hyb4_Fibro_13300853_01Oct05.tif 0" >>>> NormalizationMethod=None >>>> "NormalizationFactors=1 1" >>>> JpegImage=D:\USERS\DeSilva\asitha\TIME POINT HYBS\BRAHMA\FIBRO Vs >>>> 6HRS\non >>>> normalized results\Brahma_Hyb4_6Hrs_13300853_10Nov05_0001.jpg >>>> RatioFormulations=W1/W2 (650/546) >>>> Barcode= >>>> BackgroundSubtraction=LocalFeature >>>> "ImageOrigin=0, 0" >>>> "JpegOrigin=1610, 4830" >>>> >>>> Creator=GenePix Pro 4.0.1.17 >>>> "Scanner=Express 430529 EXP430529" >>>> FocusPosition=0 >>>> >>>> Temperature=0 >>>> LinesAveraged=1 >>>> >>>> comment= >>>> >>>> "PMTGain=0 0" >>>> >>>> "ScanPower=100 100" >>>> >>>> "LaserPower=0 0" >>>> >>>> "LaserOnTime=0 0" >>>> >>>> "ScanRegion=0,0,2200,7300" >>>> >>>> Supplier= >>>> >>>> Block Column Row Name ID X Y Dia. F650 Median >>>> F650 Mean F650 SD B650 Median B650 Mean B650 SD % > B650 1SD >>>> % > B650 2SD F650 % Sat. F546 Median F546 Mean F546 SD B546 >>>> Median B546 Mean B546 SD % > B546 1SD % > B546 2SD F546 >>> % Sat. >>>> F3 Median F3 Mean F3 SD B3 Median B3 Mean B3 SD % > B3 1SD >>>> % > B3 2SD F3 % Sat. F4 Median F4 Mean F4 SD B4 Median >>>> B4 Mean B4 SD % > B4 1SD % > B4 2SD F4 % Sat. >>> Ratio of >>>> Medians (650/546) Ratio of Means (650/546) Median of Ratios >>>> (650/546) Mean of Ratios (650/546) Ratios SD (650/546) Rgn >>>> Ratio (650/546) Rgn R? (650/546) Ratio of Medians (Ratio/2) >>>> Ratio of Means (Ratio/2) Median of Ratios (Ratio/2) Mean of >>>> Ratios (Ratio/2) Ratios SD (Ratio/2) Rgn Ratio (Ratio/2) Rgn >>>> R? (Ratio/2) Ratio of Medians (Ratio/3) Ratio of Means (Ratio/3) >>>> Median of Ratios (Ratio/3) Mean of Ratios (Ratio/3) Ratios SD >>>> (Ratio/3) Rgn Ratio (Ratio/3) Rgn R? (Ratio/3) F Pixels >>>> B Pixels Sum of Medians Sum of Means Log Ratio (650/546) Log >>>> Ratio (Ratio/2) Log Ratio (Ratio/3) F650 Median - B650 F546 >>> Median >>>> - B546 F3 Median - B3 F4 Median - B4 F650 Mean - B650 F546 >>> Mean - >>>> B546 F3 Mean - B3 F4 Mean - B4 Flags Normalize >>>> >>>> Thank you very much for your time. >>>> >>>> Hua >>>> -----Original Message----- >>>> From: Kasper Daniel Hansen [mailto:khansen at stat.Berkeley.EDU] >>>> Sent: Tuesday, December 06, 2005 1:42 PM >>>> To: Hua Weng >>>> Cc: bioconductor at stat.math.ethz.ch >>>> Subject: Re: [BioC] Error message from R2.2.0 on Linux >>>> >>>> >>>> On Dec 6, 2005, at 10:04 AM, Hua Weng wrote: >>>> >>>>> Hi, All: >>>>> >>>>> Is there anybody have problem running limma 2.2.4 from R 2.2.0 on >>>>> Linux? I can run the same code for Limma 2.2.4 from R2.2.0 on >>>>> windows without any problem. The following is information for R >>>>> and limma on linux: >>>>> >>>>>> version >>>>> _ >>>>> platform i686-pc-linux-gnu >>>>> arch i686 >>>>> os linux-gnu >>>>> system i686, linux-gnu >>>>> status >>>>> major 2 >>>>> minor 2.0 >>>>> year 2005 >>>>> month 10 >>>>> day 06 >>>>> svn rev 35749 >>>>> language R >>>>> >>>>> limma "limma" "/usr/local/R/lib/R/library" "2.4.4" >>>>> >>>>> And the following is the error message from the command: >>>>>> library(limma) >>>>>> files <- dir(pattern=".gpr") >>>>>> RG <- read.maimages(files, columns=list(R="F532 Mean", G="F635 >>>>>> Mean", Rb="B532 Median", Gb="B635 Median"), wt.fun=wtflags(0.1)) >>>>> Error in switch(source2, quantarray = { : Cannot find column >>>>> heading in image output file >>>>> In addition: Warning message: >>>>> input string 32 is invalid in this locale in: grep(pattern, x, >>>>> ignore.case, extended, value, fixed, useBytes) >>>>> >>>>> I highly appreciate any comments and suggestions for this error. >>>> >>>> Guess: you are reading in a textfile which contains non-standard >>>> characters, eg. special letters etc. You are currently operating >>>> in a >>>> local which does not support this particular character, so either >>>> change the locale or change the file. I see you are based in the US, >>>> so I am a bit surprised that you are having problems with this. >>>> >>>> Do a >>>> # env >>>> from the shell to get a list of variables, I think the locale one is >>>> called >>>> LANG >>>> (if you have variables such as LC_**** they override the LANG >>>> settings). >>>> >>>> However, I would change the file. Further debugging requires a look >>>> at the header of the file. >>>> >>>> Kasper >>>> >>>>> Hua Weng >>>>> Microarray Core Facility >>>>> 348E Noble Research Center >>>>> Department of Biochemistry and Molecular Biology >>>>> Oklahoma State University >>>>> Stillwater, OK 74078 >>>>> Phone: 405-744-6199 >>>>> Fax: 405-744-7799 >>>>> >>>>> >>>>> >>>>> >>>>> [[alternative HTML version deleted]] >>>>> >>>>> _______________________________________________ >>>>> Bioconductor mailing list >>>>> Bioconductor at stat.math.ethz.ch >>>>> https://stat.ethz.ch/mailman/listinfo/bioconductor >>>> >>> _______________________________________________ >>> Bioconductor mailing list >>> Bioconductor at stat.math.ethz.ch >>> https://stat.ethz.ch/mailman/listinfo/bioconductor >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at stat.math.ethz.ch >> https://stat.ethz.ch/mailman/listinfo/bioconductor > > > [[alternative HTML version deleted]] > > -- Dr. Jose I. de las Heras Email: J.delasHeras at ed.ac.uk The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131 6513374 Institute for Cell & Molecular Biology Fax: +44 (0)131 6507360 Swann Building, Mayfield Road University of Edinburgh Edinburgh EH9 3JR UK