print layout for Agilent data?
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Jianping Jin ▴ 890
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Dear list, When I was trying the normalizeWithinArrays function of limma I got an error: Error in switch (method, loess = {: Layout argument not specified). I was using an Agilent 4 x 44k data set and knew the Agilent printer has only one block (Row and Col having been taken already). How can I do loess normalization for each slide? Should I add a Block column into the RG, the data read-in file in order to do normalizaWithinArrays? Another question is that is there any easy way I can remove all control spots from all data files? Appreciate your help! JJ ################################## Jianping Jin Ph.D. Bioinformatics scientist Center for Bioinformatics Room 3133 Bioinformatics building CB# 7104 University of Chapel Hill Chapel Hill, NC 27599 Phone: (919)843-6105 FAX: (919)843-3103 E-Mail: jjin at email.unc.edu
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Yong Yin ▴ 60
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Thanks Yong! Your way may be a good one for "normalizeWithArrays" to work in a regular manner. But it may be questionable if you need to apply "normalizeBetweenArrays". My data set used common reference design. I may want to carry out normalization between arrays. I read in data using "read.maimages" with source="agilent". There should be some standard ways by which I can run normalization within arrays. Am I correct? best, JJ- --On Monday, October 29, 2007 2:51 PM -0500 Yong Yin <yyin at="" watson.wustl.edu=""> wrote: > If you are asking how to handle different blocks in Agilent 4x44k, we > have had the same problem. > > > In brief, we have printed the same set of 44k probes on each of the 4 > blocks, and we are applying different samples onto different blocks. > > > After discussing with our microarray facility, they find a way to > generate individual raw data file for each array using the software > coming with scanner. Then I just treat data generated from different > blocks as independent arrays. > > > I suggest you talk to the people running the machines. And I would love > to hear others' experiences. > > > Best, > > > Yong > > > ? > > > On Oct 29, 2007, at 2:19 PM, Jianping Jin wrote: > > > Dear list, > > > When I was trying the normalizeWithinArrays function of limma I got an? > error: Error in switch (method, loess = {: Layout argument not > specified).? > I was using an Agilent 4 x 44k data set and knew the Agilent printer has? > only one block (Row and Col having been taken already). How can I do > loess? > normalization for each slide? Should I add a Block column into the RG, > the? > data read-in file in order to do normalizaWithinArrays? > > > Another question is that is there any easy way I can remove all control? > spots from all data files? > > > Appreciate your help! > > > JJ > > > ################################## > Jianping Jin Ph.D. > Bioinformatics scientist > Center for Bioinformatics > Room 3133 Bioinformatics building > CB# 7104 > University of Chapel Hill > Chapel Hill, NC 27599 > Phone: (919)843-6105 > FAX: ? (919)843-3103 > E-Mail: jjin at email.unc.edu > > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > > > > > > > Yong Yin, Ph.D. > > > Senior Scientist > Genome Sequencing Center > Washington University School of Medicine,?Campus box 8501 > 4444 Forest Park > Saint Louis, MO 63108 > > > Tel: (314) 286-1415 > ################################## Jianping Jin Ph.D. Bioinformatics scientist Center for Bioinformatics Room 3133 Bioinformatics building CB# 7104 University of Chapel Hill Chapel Hill, NC 27599 Phone: (919)843-6105 FAX: (919)843-3103 E-Mail: jjin at email.unc.edu
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The Feature Extraction software from Agilent knows the format from the control file supplied by Agilent and will create 4 sets of separate files. You run your analysis as if they were singly hybridized 44K arrays. John > Thanks Yong! > > Your way may be a good one for "normalizeWithArrays" to work in a regular > manner. But it may be questionable if you need to apply > "normalizeBetweenArrays". My data set used common reference design. I may > want to carry out normalization between arrays. > > I read in data using "read.maimages" with source="agilent". There should be > some standard ways by which I can run normalization within arrays. Am I > correct? > > best, > > JJ- > > --On Monday, October 29, 2007 2:51 PM -0500 Yong Yin > <yyin at="" watson.wustl.edu=""> wrote: > >> If you are asking how to handle different blocks in Agilent 4x44k, we >> have had the same problem. >> >> >> In brief, we have printed the same set of 44k probes on each of the 4 >> blocks, and we are applying different samples onto different blocks. >> >> >> After discussing with our microarray facility, they find a way to >> generate individual raw data file for each array using the software >> coming with scanner. Then I just treat data generated from different >> blocks as independent arrays. >> >> >> I suggest you talk to the people running the machines. And I would love >> to hear others' experiences. >> >> >> Best, >> >> >> Yong >> >> >> ? >> >> >> On Oct 29, 2007, at 2:19 PM, Jianping Jin wrote: >> >> >> Dear list, >> >> >> When I was trying the normalizeWithinArrays function of limma I got an? >> error: Error in switch (method, loess = {: Layout argument not >> specified).? >> I was using an Agilent 4 x 44k data set and knew the Agilent printer has? >> only one block (Row and Col having been taken already). How can I do >> loess? >> normalization for each slide? Should I add a Block column into the RG, >> the? >> data read-in file in order to do normalizaWithinArrays? >> >> >> Another question is that is there any easy way I can remove all control? >> spots from all data files? >> >> >> Appreciate your help! >> >> >> JJ >> >> >> ################################## >> Jianping Jin Ph.D. >> Bioinformatics scientist >> Center for Bioinformatics >> Room 3133 Bioinformatics building >> CB# 7104 >> University of Chapel Hill >> Chapel Hill, NC 27599 >> Phone: (919)843-6105 >> FAX: ? (919)843-3103 >> E-Mail: jjin at email.unc.edu >> >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at stat.math.ethz.ch >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor >> >> >> >> >> >> >> Yong Yin, Ph.D. >> >> >> Senior Scientist >> Genome Sequencing Center >> Washington University School of Medicine,?Campus box 8501 >> 4444 Forest Park >> Saint Louis, MO 63108 >> >> >> Tel: (314) 286-1415 >> > > > > ################################## > Jianping Jin Ph.D. > Bioinformatics scientist > Center for Bioinformatics > Room 3133 Bioinformatics building > CB# 7104 > University of Chapel Hill > Chapel Hill, NC 27599 > Phone: (919)843-6105 > FAX: (919)843-3103 > E-Mail: jjin at email.unc.edu > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor
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Hi John, This is my first to handle the Agilent 4x44k arrays. Yes you are right. My collaborator ran 9 chips and sent me 36 separate files. For "read.maimages" they should serve as individual data files to read in just like regular arrays. Am I right? Is there any other file(s) which need to be read in? best, JP- --On Monday, October 29, 2007 5:26 PM -0700 John Fernandes <jfernand at="" stanford.edu=""> wrote: > The Feature Extraction software from Agilent knows the format from the > control file supplied by Agilent and will create 4 sets of separate > files. You run your analysis as if they were singly hybridized 44K > arrays. > > John > > >> Thanks Yong! >> >> Your way may be a good one for "normalizeWithArrays" to work in a regular >> manner. But it may be questionable if you need to apply >> "normalizeBetweenArrays". My data set used common reference design. I may >> want to carry out normalization between arrays. >> >> I read in data using "read.maimages" with source="agilent". There should >> be some standard ways by which I can run normalization within arrays. Am >> I correct? >> >> best, >> >> JJ- >> >> --On Monday, October 29, 2007 2:51 PM -0500 Yong Yin >> <yyin at="" watson.wustl.edu=""> wrote: >> >>> If you are asking how to handle different blocks in Agilent 4x44k, we >>> have had the same problem. >>> >>> >>> In brief, we have printed the same set of 44k probes on each of the 4 >>> blocks, and we are applying different samples onto different blocks. >>> >>> >>> After discussing with our microarray facility, they find a way to >>> generate individual raw data file for each array using the software >>> coming with scanner. Then I just treat data generated from different >>> blocks as independent arrays. >>> >>> >>> I suggest you talk to the people running the machines. And I would love >>> to hear others' experiences. >>> >>> >>> Best, >>> >>> >>> Yong >>> >>> >>> ? >>> >>> >>> On Oct 29, 2007, at 2:19 PM, Jianping Jin wrote: >>> >>> >>> Dear list, >>> >>> >>> When I was trying the normalizeWithinArrays function of limma I got an? >>> error: Error in switch (method, loess = {: Layout argument not >>> specified).? >>> I was using an Agilent 4 x 44k data set and knew the Agilent printer >>> has? only one block (Row and Col having been taken already). How can I >>> do loess? >>> normalization for each slide? Should I add a Block column into the RG, >>> the? >>> data read-in file in order to do normalizaWithinArrays? >>> >>> >>> Another question is that is there any easy way I can remove all >>> control? spots from all data files? >>> >>> >>> Appreciate your help! >>> >>> >>> JJ >>> >>> >>> ################################## >>> Jianping Jin Ph.D. >>> Bioinformatics scientist >>> Center for Bioinformatics >>> Room 3133 Bioinformatics building >>> CB# 7104 >>> University of Chapel Hill >>> Chapel Hill, NC 27599 >>> Phone: (919)843-6105 >>> FAX: ? (919)843-3103 >>> E-Mail: jjin at email.unc.edu >>> >>> >>> _______________________________________________ >>> Bioconductor mailing list >>> Bioconductor at stat.math.ethz.ch >>> https://stat.ethz.ch/mailman/listinfo/bioconductor >>> Search the archives: >>> http://news.gmane.org/gmane.science.biology.informatics.conductor >>> >>> >>> >>> >>> >>> >>> Yong Yin, Ph.D. >>> >>> >>> Senior Scientist >>> Genome Sequencing Center >>> Washington University School of Medicine,?Campus box 8501 >>> 4444 Forest Park >>> Saint Louis, MO 63108 >>> >>> >>> Tel: (314) 286-1415 >>> >> >> >> >> ################################## >> Jianping Jin Ph.D. >> Bioinformatics scientist >> Center for Bioinformatics >> Room 3133 Bioinformatics building >> CB# 7104 >> University of Chapel Hill >> Chapel Hill, NC 27599 >> Phone: (919)843-6105 >> FAX: (919)843-3103 >> E-Mail: jjin at email.unc.edu >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at stat.math.ethz.ch >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor ################################## Jianping Jin Ph.D. Bioinformatics scientist Center for Bioinformatics Room 3133 Bioinformatics building CB# 7104, Campus Phone: (919)843-6105 FAX: (919)843-3103 E-Mail: jjin at unc.edu
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Jianping Jin wrote: > Hi John, > > This is my first to handle the Agilent 4x44k arrays. Yes you are right. My > collaborator ran 9 chips and sent me 36 separate files. For "read.maimages" > they should serve as individual data files to read in just like regular > arrays. Am I right? Is there any other file(s) which need to be read in? That should do it. Sean
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Thanks Sean. Here back to my original questions. Reading in data with "read.maimages" had no problems (see below) > RG$genes[1:5,] Row Col Start Sequence ProbeUID ControlType ProbeName GeneName 1 1 1 0 0 1 GE_BrightCorner GE_BrightCorner 2 1 2 0 1 1 DarkCorner DarkCorner 3 1 3 0 1 1 DarkCorner DarkCorner 4 1 4 0 1 1 DarkCorner DarkCorner 5 1 5 0 1 1 DarkCorner DarkCorner SystematicName Description 1 GE_BrightCorner 2 DarkCorner 3 DarkCorner 4 DarkCorner 5 DarkCorner But when I was trying the normalizeWithinArrays function I got an error: Error in switch (method, loess= {: Layout argument not specified). As you can see the RG file has already taken in the Row and Col information. There is no Block column however, as Agilent array has not designed as different blocks. That may be the reason of the error. I wanted to know how people handle this situation in order to carry out loess normalization for each array. Do we need to add a "Block" column into RG$genes or specify gene layout format? best, JJ- --On Tuesday, October 30, 2007 10:12 AM -0400 Sean Davis <sdavis2 at="" mail.nih.gov=""> wrote: > Jianping Jin wrote: >> Hi John, >> >> This is my first to handle the Agilent 4x44k arrays. Yes you are right. >> My collaborator ran 9 chips and sent me 36 separate files. For >> "read.maimages" they should serve as individual data files to read in >> just like regular arrays. Am I right? Is there any other file(s) which >> need to be read in? > > That should do it. > > Sean ################################## Jianping Jin Ph.D. Bioinformatics scientist Center for Bioinformatics Room 3133 Bioinformatics building CB# 7104 University of Chapel Hill Chapel Hill, NC 27599 Phone: (919)843-6105 FAX: (919)843-3103 E-Mail: jjin at email.unc.edu
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Hi Yong, It is good to know that your files do have the Block column. I have never gotten that column in Agilent FT output files no matter of versions of 8.x. or 9.x as far as I can remember. I need to check with our core facility for that. BTW Sean's suggestion worked well for me. Thanks a lot for your help! JJ- --On Tuesday, October 30, 2007 10:24 AM -0500 Yong Yin <yyin at="" watson.wustl.edu=""> wrote: > The block-level files I received are indeed txt files. When I open them > by a txt editor just to check, it has the block info as the first column. > > > After I inputed them with a target.ext file, the block info is still > there. Such as: > > > >> RGH$genes[1:5,] > ??Block Row Column ? ? ? ? ? ? ?ID ? ? ? ? ? ?Name > 1 ? ? 1 ? 1 ? ? ?1 GE_BrightCorner GE_BrightCorner > 2 ? ? 1 ? 1 ? ? ?2 GE_BrightCorner GE_BrightCorner > 3 ? ? 1 ? 1 ? ? ?3 ? ? ?DarkCorner ? ? ?DarkCorner > 4 ? ? 1 ? 1 ? ? ?4 ? ? ?DarkCorner ? ? ?DarkCorner > 5 ? ? 1 ? 1 ? ? ?5 ? ? ?DarkCorner ? ? ?DarkCorner > > > So, is it possible your files are not complete? > > For normalization, I used the same command as Sean suggested: > > > > MAHWC<-normalizeWithinArrays(RGHWC, method="loess") > > > It worked just fine. > > > Yong > > > > > > On Oct 30, 2007, at 9:50 AM, Jianping Jin wrote: > > > Thanks Sean. > > > Here back to my original questions. Reading in data with "read.maimages"? > had no problems (see below) > > > > > RG$genes[1:5,] > > > ? Row Col Start Sequence ProbeUID ControlType ? ? ? ProbeName? > GeneName > 1 ? 1 ? 1 ? ? 0 ? ? ? ? ? ? ? ? 0 ? ? ? ? ? 1 > GE_BrightCorner? > GE_BrightCorner > 2 ? 1 ? 2 ? ? 0 ? ? ? ? ? ? ? ? 1 ? ? ? ? ? 1? ? ? > DarkCorner? > DarkCorner > 3 ? 1 ? 3 ? ? 0 ? ? ? ? ? ? ? ? 1 ? ? ? ? ? 1? ? ? > DarkCorner? > DarkCorner > 4 ? 1 ? 4 ? ? 0 ? ? ? ? ? ? ? ? 1 ? ? ? ? ? 1? ? ? > DarkCorner? > DarkCorner > 5 ? 1 ? 5 ? ? 0 ? ? ? ? ? ? ? ? 1 ? ? ? ? ? 1? ? ? > DarkCorner? > DarkCorner > ?? SystematicName Description > 1 GE_BrightCorner > 2? ? ? DarkCorner > 3? ? ? DarkCorner > 4? ? ? DarkCorner > 5? ? ? DarkCorner > > > But when I was trying the normalizeWithinArrays function I got an error: > Error in switch (method, loess= {: Layout argument not specified). > > > As you can see the RG file has already taken in the Row and Col? > information. There is no Block column however, as Agilent array has not? > designed as different blocks. That may be the reason of the error. I > wanted? > to know how people handle this situation in order to carry out loess? > normalization for each array. Do we need to add a "Block" column into? > RG$genes or specify gene layout format? > > > best, > > > JJ- > > > --On Tuesday, October 30, 2007 10:12 AM -0400 Sean Davis? > <sdavis2 at="" mail.nih.gov=""> wrote: > > > > > Jianping Jin wrote: > > > Hi John, > > > This is my first to handle the Agilent 4x44k arrays. Yes you are right. > My? collaborator ran 9 chips and sent me 36 separate files. For > "read.maimages"? they should serve as individual data files to read in > just like regular? arrays. Am I right? Is there any other file(s) which > need to be read in? > > > > > That should do it. > > > Sean > > > > > > > > > ################################## > Jianping Jin Ph.D. > Bioinformatics scientist > Center for Bioinformatics > Room 3133 Bioinformatics building > CB# 7104 > University of Chapel Hill > Chapel Hill, NC 27599 > Phone: (919)843-6105 > FAX: ? (919)843-3103 > E-Mail: jjin at email.unc.edu > > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > > > > > > > Yong Yin, Ph.D. > > > Senior Scientist > Genome Sequencing Center > Washington University School of Medicine,?Campus box 8501 > 4444 Forest Park > Saint Louis, MO 63108 > > > Tel: (314) 286-1415 > ################################## Jianping Jin Ph.D. Bioinformatics scientist Center for Bioinformatics Room 3133 Bioinformatics building CB# 7104 University of Chapel Hill Chapel Hill, NC 27599 Phone: (919)843-6105 FAX: (919)843-3103 E-Mail: jjin at email.unc.edu
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Jianping Jin wrote: > Thanks Sean. > > Here back to my original questions. Reading in data with "read.maimages" > had no problems (see below) > >> RG$genes[1:5,] > Row Col Start Sequence ProbeUID ControlType ProbeName GeneName > 1 1 1 0 0 1 GE_BrightCorner > GE_BrightCorner > 2 1 2 0 1 1 DarkCorner DarkCorner > 3 1 3 0 1 1 DarkCorner DarkCorner > 4 1 4 0 1 1 DarkCorner DarkCorner > 5 1 5 0 1 1 DarkCorner DarkCorner > SystematicName Description > 1 GE_BrightCorner > 2 DarkCorner > 3 DarkCorner > 4 DarkCorner > 5 DarkCorner > > But when I was trying the normalizeWithinArrays function I got an error: > Error in switch (method, loess= {: Layout argument not specified). Reading the help for normalizeWithinArrays, you might notice that the default method is "printtiploess". That is fine, except that you need to specify the layout by hand. However, Agilent does not use "print tips" or blocks, so printtiploess is not appropriate for Agilent arrays. I think if you specify method="loess", you will probably find that things work just fine. Sean
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Great Sean! You solved my problem. Do you know an easy way to remove all Agilent control spots, including empty, negative and positive controls from a gene list to be analyzed. best, JJ- --On Tuesday, October 30, 2007 10:54 AM -0400 Sean Davis <sdavis2 at="" mail.nih.gov=""> wrote: > Jianping Jin wrote: >> Thanks Sean. >> >> Here back to my original questions. Reading in data with "read.maimages" >> had no problems (see below) >> >>> RG$genes[1:5,] >> Row Col Start Sequence ProbeUID ControlType ProbeName GeneName >> 1 1 1 0 0 1 GE_BrightCorner >> GE_BrightCorner >> 2 1 2 0 1 1 DarkCorner DarkCorner >> 3 1 3 0 1 1 DarkCorner DarkCorner >> 4 1 4 0 1 1 DarkCorner DarkCorner >> 5 1 5 0 1 1 DarkCorner DarkCorner >> SystematicName Description >> 1 GE_BrightCorner >> 2 DarkCorner >> 3 DarkCorner >> 4 DarkCorner >> 5 DarkCorner >> >> But when I was trying the normalizeWithinArrays function I got an error: >> Error in switch (method, loess= {: Layout argument not specified). > > Reading the help for normalizeWithinArrays, you might notice that the > default method is "printtiploess". That is fine, except that you need > to specify the layout by hand. However, Agilent does not use "print > tips" or blocks, so printtiploess is not appropriate for Agilent arrays. > I think if you specify method="loess", you will probably find that > things work just fine. > > Sean ################################## Jianping Jin Ph.D. Bioinformatics scientist Center for Bioinformatics Room 3133 Bioinformatics building CB# 7104, Campus Phone: (919)843-6105 FAX: (919)843-3103 E-Mail: jjin at unc.edu
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Jianping Jin wrote: > Great Sean! You solved my problem. > > Do you know an easy way to remove all Agilent control spots, including > empty, negative and positive controls from a gene list to be analyzed. I believe the controltype column contains these information. You can just remove probes with controltype!=0, if I recall correctly. > --On Tuesday, October 30, 2007 10:54 AM -0400 Sean Davis > <sdavis2 at="" mail.nih.gov=""> wrote: > >> Jianping Jin wrote: >>> Thanks Sean. >>> >>> Here back to my original questions. Reading in data with "read.maimages" >>> had no problems (see below) >>> >>>> RG$genes[1:5,] >>> Row Col Start Sequence ProbeUID ControlType ProbeName GeneName >>> 1 1 1 0 0 1 GE_BrightCorner >>> GE_BrightCorner >>> 2 1 2 0 1 1 DarkCorner DarkCorner >>> 3 1 3 0 1 1 DarkCorner DarkCorner >>> 4 1 4 0 1 1 DarkCorner DarkCorner >>> 5 1 5 0 1 1 DarkCorner DarkCorner >>> SystematicName Description >>> 1 GE_BrightCorner >>> 2 DarkCorner >>> 3 DarkCorner >>> 4 DarkCorner >>> 5 DarkCorner >>> >>> But when I was trying the normalizeWithinArrays function I got an error: >>> Error in switch (method, loess= {: Layout argument not specified). >> >> Reading the help for normalizeWithinArrays, you might notice that the >> default method is "printtiploess". That is fine, except that you need >> to specify the layout by hand. However, Agilent does not use "print >> tips" or blocks, so printtiploess is not appropriate for Agilent arrays. >> I think if you specify method="loess", you will probably find that >> things work just fine. >> >> Sean > > > > ################################## > Jianping Jin Ph.D. > Bioinformatics scientist > Center for Bioinformatics > Room 3133 Bioinformatics building > CB# 7104, Campus > Phone: (919)843-6105 > FAX: (919)843-3103 > E-Mail: jjin at unc.edu
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