Deciding on a cut off after QC
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Ankit Pal ▴ 230
@ankit-pal-1242
Last seen 10.2 years ago
Dear All, I'm using LIMMA to analyse a set of GPR files. I used the weight fuction to apply QC parameter threshold values recommended by Genepix. The code for the same is >myfun <- function(x,threshold=55){ + okred <- abs(x[,"% > B635+2SD"]) < threshold + okgreen <- abs(x[,"% > B532+2SD"]) < threshold + as.numeric(okgreen & okred) } On completion of the analysis, all the spots showed up in the results file inspite of being flagged off. I understand that on being flagged off by limma (wt = 0), the spots are not considered for further analysis. Is there any way they can be excluded from the final result file. Also, if I get an output of all the spots (38000 in my case) how do I decide on a cut off. Do I use the rank or something else? Waiting eagerly for a reply, thank you, -Ankit Stay connected, organized, and protected. Take the tour:
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@gordon-smyth
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WEHI, Melbourne, Australia
> Date: Sun, 15 May 2005 21:34:18 -0700 (PDT) > From: Ankit Pal <pal_ankit2000@yahoo.com> > Subject: [BioC] Deciding on a cut off after QC > To: bioconductor@stat.math.ethz.ch > > Dear All, > I'm using LIMMA to analyse a set of GPR files. > I used the weight fuction to apply QC parameter > threshold values recommended by Genepix. > The code for the same is > >>myfun <- function(x,threshold=55){ > + okred <- abs(x[,"% > B635+2SD"]) < threshold > + okgreen <- abs(x[,"% > B532+2SD"]) < threshold > + as.numeric(okgreen & okred) > } > > On completion of the analysis, all the spots showed > up in the results file inspite of being flagged off. I > understand that on being flagged off by limma (wt = > 0), the spots are not considered for further analysis. > Is there any way they can be excluded from the final > result file. What result file? > Also, if I get an output of all the spots (38000 in my > case) how do I decide on a cut off. Do I use the rank > or something else? What output? Gordon > Waiting eagerly for a reply, > thank you, > -Ankit
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Ankit Pal ▴ 230
@ankit-pal-1242
Last seen 10.2 years ago
Dear Dr Smyth, I'm sorry for not having specified which result file. It is the final result summary we get after we give the command Resultfile <- topTable(fit,n=200, adjust="fdr") A sample result file has been attached. The code I used for my analysis is > targets <- readTargets("target.txt") #The QC filter > myfun <- function(x,threshold=55){ + okred <- abs(x[,"% > B635+2SD"]) > threshold + okgreen <- abs(x[,"% > B532+2SD"]) > threshold + okflag <- abs(x[,"Flags"]) > 0 + okRGN <- abs(x[,"Rgn R˛"]) > 0.6 + as.numeric(okgreen || okred || okflag || okRGN) + } #end of QC filter > RG_7 <- read.maimages(targets$FileName, source="genepix",wt.fun=myfun) > RG_7$genes <- readGAL() > RG_7$printer <- getLayout(RG_7$genes) > MA_7 <- normalizeWithinArrays(RG_7,method="loess") > MA_7 <- normalizeBetweenArrays(MA_7) > fit_7 <- lmFit(MA_7, design=c(1,-1,1,-1)) > fit_7 <- eBayes(fit_7) > options(digits=3) > Resultfile_7 <- topTable(fit_7, n=39000, adjust="fdr") > Resdat_7 <-data.frame(Resultfile_7) > write.table(Resdat_,file='Result.csv',quote = FALSE, sep = "\t") I understand that the spots that do not qualify the QC filter are given a weight of "0" by limma and are not considered for normalization and will not affect the analysis. The result file I get contains all the spots (38000) in my case. Didn't the spots that were bad get removed from the final result? If not what is the cut off value (B, p etc) that I need to use to get a set of reliable spots(I cant use all the 38000) from my result file for my analysis. Is there a fixed formula to derive the same as the values vary with the analysis. Waiting for your reply, Thank you, -Ankit --- Gordon K Smyth <smyth@wehi.edu.au> wrote: > > Date: Sun, 15 May 2005 21:34:18 -0700 (PDT) > > From: Ankit Pal <pal_ankit2000@yahoo.com> > > Subject: [BioC] Deciding on a cut off after QC > > To: bioconductor@stat.math.ethz.ch > > > > Dear All, > > I'm using LIMMA to analyse a set of GPR files. > > I used the weight fuction to apply QC parameter > > threshold values recommended by Genepix. > > The code for the same is > > > >>myfun <- function(x,threshold=55){ > > + okred <- abs(x[,"% > B635+2SD"]) < threshold > > + okgreen <- abs(x[,"% > B532+2SD"]) < threshold > > + as.numeric(okgreen & okred) > > } > > > > On completion of the analysis, all the spots > showed > > up in the results file inspite of being flagged > off. I > > understand that on being flagged off by limma (wt > = > > 0), the spots are not considered for further > analysis. > > Is there any way they can be excluded from the > final > > result file. > > What result file? > > > Also, if I get an output of all the spots (38000 > in my > > case) how do I decide on a cut off. Do I use the > rank > > or something else? > > What output? > > Gordon > > > Waiting eagerly for a reply, > > thank you, > > -Ankit > > > Stay connected, organized, and protected. 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On Mon, May 16, 2005 9:49 pm, Ankit Pal said: > Dear Dr Smyth, > I'm sorry for not having specified which result file. > It is the final result summary we get after we give > the command > Resultfile <- topTable(fit,n=200, adjust="fdr") > A sample result file has been attached. > The code I used for my analysis is > >> targets <- readTargets("target.txt") > > #The QC filter >> myfun <- function(x,threshold=55){ > + okred <- abs(x[,"% > B635+2SD"]) > threshold > + okgreen <- abs(x[,"% > B532+2SD"]) > threshold > + okflag <- abs(x[,"Flags"]) > 0 > + okRGN <- abs(x[,"Rgn R?"]) > 0.6 > + as.numeric(okgreen || okred || okflag || okRGN) > + } > #end of QC filter > >> RG_7 <- read.maimages(targets$FileName, > source="genepix",wt.fun=myfun) >> RG_7$genes <- readGAL() As I said last week, this command is not needed with GenePix data. You should omit it. >> RG_7$printer <- getLayout(RG_7$genes) >> MA_7 <- normalizeWithinArrays(RG_7,method="loess") >> MA_7 <- normalizeBetweenArrays(MA_7) >> fit_7 <- lmFit(MA_7, design=c(1,-1,1,-1)) >> fit_7 <- eBayes(fit_7) >> options(digits=3) >> Resultfile_7 <- topTable(fit_7, n=39000, > adjust="fdr") >> Resdat_7 <-data.frame(Resultfile_7) Resultfile_7 is already a data.frame. >> write.table(Resdat_,file='Result.csv',quote = FALSE, > sep = "\t") > > I understand that the spots that do not qualify the QC > filter are given a weight of "0" by limma and are not > considered for normalization and will not affect the > analysis. > The result file I get contains all the spots (38000) > in my case. Mmm, this doesn't make sense. You have 4 arrays with 39000 spots on each array. Hence you have 4*39000 spots, not 39000. The output from topTable() gives a summarized log-ratio for each probe, not a result for each spot. Gordon > Didn't the spots that were bad get removed from the > final result? > If not what is the cut off value (B, p etc) that I > need to use to get a set of reliable spots(I cant use > all the 38000) from my result file for my analysis. > Is there a fixed formula to derive the same as the > values vary with the analysis. > Waiting for your reply, > Thank you, > -Ankit
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Ankit Pal ▴ 230
@ankit-pal-1242
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Dear Dr Smyth, I guess there has been a mistake in communication. There are 39000 spots on the array but the number of probes are lesser as there are replicates(not a fixed number) on the array. The output from toptable() gives me a total of 39000 rows, one for each spot. I need to give an output of the spots most significant in this list. What is the cut off value(p or B or any other) I need to use above which I can consider a set of differentially expressed genes significant enough for further use (eg :function based clustering of all significantly overexpressed genes). I cannot use all the genes in the topTable() output. Where do I put the cut off? Is there a way I can calculate it? sincerely, -Ankit --- Gordon K Smyth <smyth@wehi.edu.au> wrote: > On Mon, May 16, 2005 9:49 pm, Ankit Pal said: > > Dear Dr Smyth, > > I'm sorry for not having specified which result > file. > > It is the final result summary we get after we > give > > the command > > Resultfile <- topTable(fit,n=200, adjust="fdr") > > A sample result file has been attached. > > The code I used for my analysis is > > > >> targets <- readTargets("target.txt") > > > > #The QC filter > >> myfun <- function(x,threshold=55){ > > + okred <- abs(x[,"% > B635+2SD"]) > threshold > > + okgreen <- abs(x[,"% > B532+2SD"]) > threshold > > + okflag <- abs(x[,"Flags"]) > 0 > > + okRGN <- abs(x[,"Rgn R˛"]) > 0.6 > > + as.numeric(okgreen || okred || okflag || okRGN) > > + } > > #end of QC filter > > > >> RG_7 <- read.maimages(targets$FileName, > > source="genepix",wt.fun=myfun) > >> RG_7$genes <- readGAL() > > As I said last week, this command is not needed with > GenePix data. You should omit it. > > >> RG_7$printer <- getLayout(RG_7$genes) > >> MA_7 <- > normalizeWithinArrays(RG_7,method="loess") > >> MA_7 <- normalizeBetweenArrays(MA_7) > >> fit_7 <- lmFit(MA_7, design=c(1,-1,1,-1)) > >> fit_7 <- eBayes(fit_7) > >> options(digits=3) > >> Resultfile_7 <- topTable(fit_7, n=39000, > > adjust="fdr") > >> Resdat_7 <-data.frame(Resultfile_7) > > Resultfile_7 is already a data.frame. > > >> write.table(Resdat_,file='Result.csv',quote = > FALSE, > > sep = "\t") > > > > I understand that the spots that do not qualify > the QC > > filter are given a weight of "0" by limma and are > not > > considered for normalization and will not affect > the > > analysis. > > The result file I get contains all the spots > (38000) > > in my case. > > Mmm, this doesn't make sense. You have 4 arrays > with 39000 spots on each array. Hence you have > 4*39000 spots, not 39000. The output from > topTable() gives a summarized log-ratio for each > probe, > not a result for each spot. > > Gordon > > > Didn't the spots that were bad get removed from > the > > final result? > > If not what is the cut off value (B, p etc) that I > > need to use to get a set of reliable spots(I cant > use > > all the 38000) from my result file for my > analysis. > > Is there a fixed formula to derive the same as the > > values vary with the analysis. > > Waiting for your reply, > > Thank you, > > -Ankit > > >
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See comments below. On Mon, 2005-05-16 at 05:42 -0700, Ankit Pal wrote: > Dear Dr Smyth, > I guess there has been a mistake in communication. > There are 39000 spots on the array but the number of > probes are lesser as there are replicates(not a fixed > number) on the array. > The output from toptable() gives me a total of 39000 > rows, one for each spot. > I need to give an output of the spots most significant > in this list. I do not understand this clearly. Do you mean you have some duplicated spots or is there redundancy when you convert to something common like unigene ID ? If the former, I believe there are some facilities within LIMMA for equal number duplications per spots but I am not sure. For the latter, you could use tapply or something to find the min/max of the statistics. > What is the cut off value(p or B or any other) I need > to use above which I can consider a set of > differentially expressed genes significant enough for > further use (eg :function based clustering of all > significantly overexpressed genes). > I cannot use all the genes in the topTable() output. > Where do I put the cut off? > Is there a way I can calculate it? Selection of p-value threshold is arbitrary. However it would be a good idea to adjust for multiple comparison. My preferred method is the FDR by Benjamini and Hochberg but there are many other methods too each with a different meaning. I will illustrate FDR using the slightly modified example from help(topTable) : set.seed(123) # for reproducibility only - do not use this in real life M <- matrix(rnorm(10000*6,sd=0.3), 10000, 6) M[1:100, 1:3] <- M[1:100, 1:3] + 2 design <- cbind( First3Arrays=c(1,1,1,0,0,0), Last3Arrays=c(0,0,0,1,1,1)) fit <- lmFit(M, design=design) fit <- eBayes(fit) tab <- topTable( fit, adjust.method="fdr", sort.by="p", n=nrow(M) ) positives <- rownames(tab)[ which(tab[ , "P.Value"] < 0.05) ] The "positives" are the names of genes that we declare as differentially expression and it is expected that 5% of these genes are false positives on average. Note that in this case we can explicitly calculate the FP rate as follows because we have simulated the datasets. mean( ifelse( as.numeric(positives) <= 100, 0, 1 ) ) 0.06542056 I am sure Gordon Symth has a far more elegant and flexible version of this documented somewhere in his user guide. Regards, Adai > sincerely, > -Ankit > > --- Gordon K Smyth <smyth@wehi.edu.au> wrote: > > On Mon, May 16, 2005 9:49 pm, Ankit Pal said: > > > Dear Dr Smyth, > > > I'm sorry for not having specified which result > > file. > > > It is the final result summary we get after we > > give > > > the command > > > Resultfile <- topTable(fit,n=200, adjust="fdr") > > > A sample result file has been attached. > > > The code I used for my analysis is > > > > > >> targets <- readTargets("target.txt") > > > > > > #The QC filter > > >> myfun <- function(x,threshold=55){ > > > + okred <- abs(x[,"% > B635+2SD"]) > threshold > > > + okgreen <- abs(x[,"% > B532+2SD"]) > threshold > > > + okflag <- abs(x[,"Flags"]) > 0 > > > + okRGN <- abs(x[,"Rgn R"]) > 0.6 > > > + as.numeric(okgreen || okred || okflag || okRGN) > > > + } > > > #end of QC filter > > > > > >> RG_7 <- read.maimages(targets$FileName, > > > source="genepix",wt.fun=myfun) > > >> RG_7$genes <- readGAL() > > > > As I said last week, this command is not needed with > > GenePix data. You should omit it. > > > > >> RG_7$printer <- getLayout(RG_7$genes) > > >> MA_7 <- > > normalizeWithinArrays(RG_7,method="loess") > > >> MA_7 <- normalizeBetweenArrays(MA_7) > > >> fit_7 <- lmFit(MA_7, design=c(1,-1,1,-1)) > > >> fit_7 <- eBayes(fit_7) > > >> options(digits=3) > > >> Resultfile_7 <- topTable(fit_7, n=39000, > > > adjust="fdr") > > >> Resdat_7 <-data.frame(Resultfile_7) > > > > Resultfile_7 is already a data.frame. > > > > >> write.table(Resdat_,file='Result.csv',quote = > > FALSE, > > > sep = "\t") > > > > > > I understand that the spots that do not qualify > > the QC > > > filter are given a weight of "0" by limma and are > > not > > > considered for normalization and will not affect > > the > > > analysis. > > > The result file I get contains all the spots > > (38000) > > > in my case. > > > > Mmm, this doesn't make sense. You have 4 arrays > > with 39000 spots on each array. Hence you have > > 4*39000 spots, not 39000. The output from > > topTable() gives a summarized log-ratio for each > > probe, > > not a result for each spot. > > > > Gordon > > > > > Didn't the spots that were bad get removed from > > the > > > final result? > > > If not what is the cut off value (B, p etc) that I > > > need to use to get a set of reliable spots(I cant > > use > > > all the 38000) from my result file for my > > analysis. > > > Is there a fixed formula to derive the same as the > > > values vary with the analysis. > > > Waiting for your reply, > > > Thank you, > > > -Ankit > > > > > > > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor >
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Dear Adai, Thank you for the detailed response. Correct me if I'm wrong. What you are saying is that after applying the "fdr" I give a cut off of 0.05 for the p-value and write all those spots into an object (positives). That means, I do not consider the B values. In essence, it is just a one sample t test afetr adjusting the p-value and taking into consideration all those spots that have a p-value < 0.05? >From my code in a previous mail to Gordon Smyth, I have done a quality control (QC) using parameters and threshold values prescribed by genepix. I know from experience, a large number of spots do not get through the filter. As I understand from LIMMA, a weight of "0" is given to any spot that does not get through the QC filter. How do I exclude these spots from the final result summary file? I need to get a set of significantly differentially expressed genes that have got through the QC filter. How do I do it using LIMMA? Thank you, -Ankit --- Adaikalavan Ramasamy <ramasamy@cancer.org.uk> wrote: > See comments below. > > On Mon, 2005-05-16 at 05:42 -0700, Ankit Pal wrote: > > Dear Dr Smyth, > > I guess there has been a mistake in communication. > > There are 39000 spots on the array but the number > of > > probes are lesser as there are replicates(not a > fixed > > number) on the array. > > The output from toptable() gives me a total of > 39000 > > rows, one for each spot. > > I need to give an output of the spots most > significant > > in this list. > > I do not understand this clearly. Do you mean you > have some duplicated > spots or is there redundancy when you convert to > something common like > unigene ID ? > > If the former, I believe there are some facilities > within LIMMA for > equal number duplications per spots but I am not > sure. For the latter, > you could use tapply or something to find the > min/max of the statistics. > > > What is the cut off value(p or B or any other) I > need > > to use above which I can consider a set of > > differentially expressed genes significant enough > for > > further use (eg :function based clustering of all > > significantly overexpressed genes). > > I cannot use all the genes in the topTable() > output. > > Where do I put the cut off? > > Is there a way I can calculate it? > > Selection of p-value threshold is arbitrary. However > it would be a good > idea to adjust for multiple comparison. My preferred > method is the FDR > by Benjamini and Hochberg but there are many other > methods too each with > a different meaning. I will illustrate FDR using the > slightly modified > example from help(topTable) : > > set.seed(123) # for reproducibility only - do not > use this in real life > > M <- matrix(rnorm(10000*6,sd=0.3), 10000, 6) > M[1:100, 1:3] <- M[1:100, 1:3] + 2 > design <- cbind( First3Arrays=c(1,1,1,0,0,0), > Last3Arrays=c(0,0,0,1,1,1)) > fit <- lmFit(M, design=design) > fit <- eBayes(fit) > > tab <- topTable( fit, adjust.method="fdr", > sort.by="p", n=nrow(M) ) > > positives <- rownames(tab)[ which(tab[ , "P.Value"] > < 0.05) ] > > The "positives" are the names of genes that we > declare as differentially > expression and it is expected that 5% of these genes > are false positives > on average. > > Note that in this case we can explicitly calculate > the FP rate as > follows because we have simulated the datasets. > > mean( ifelse( as.numeric(positives) <= 100, 0, 1 ) ) > 0.06542056 > > I am sure Gordon Symth has a far more elegant and > flexible version of > this documented somewhere in his user guide. > > Regards, Adai > > > > sincerely, > > -Ankit > > > > --- Gordon K Smyth <smyth@wehi.edu.au> wrote: > > > On Mon, May 16, 2005 9:49 pm, Ankit Pal said: > > > > Dear Dr Smyth, > > > > I'm sorry for not having specified which > result > > > file. > > > > It is the final result summary we get after we > > > give > > > > the command > > > > Resultfile <- topTable(fit,n=200, > adjust="fdr") > > > > A sample result file has been attached. > > > > The code I used for my analysis is > > > > > > > >> targets <- readTargets("target.txt") > > > > > > > > #The QC filter > > > >> myfun <- function(x,threshold=55){ > > > > + okred <- abs(x[,"% > B635+2SD"]) > threshold > > > > + okgreen <- abs(x[,"% > B532+2SD"]) > > threshold > > > > + okflag <- abs(x[,"Flags"]) > 0 > > > > + okRGN <- abs(x[,"Rgn R"]) > 0.6 > > > > + as.numeric(okgreen || okred || okflag || > okRGN) > > > > + } > > > > #end of QC filter > > > > > > > >> RG_7 <- read.maimages(targets$FileName, > > > > source="genepix",wt.fun=myfun) > > > >> RG_7$genes <- readGAL() > > > > > > As I said last week, this command is not needed > with > > > GenePix data. You should omit it. > > > > > > >> RG_7$printer <- getLayout(RG_7$genes) > > > >> MA_7 <- > > > normalizeWithinArrays(RG_7,method="loess") > > > >> MA_7 <- normalizeBetweenArrays(MA_7) > > > >> fit_7 <- lmFit(MA_7, design=c(1,-1,1,-1)) > > > >> fit_7 <- eBayes(fit_7) > > > >> options(digits=3) > > > >> Resultfile_7 <- topTable(fit_7, n=39000, > > > > adjust="fdr") > > > >> Resdat_7 <-data.frame(Resultfile_7) > > > > > > Resultfile_7 is already a data.frame. > > > > > > >> write.table(Resdat_,file='Result.csv',quote = > > > FALSE, > > > > sep = "\t") > > > > > > > > I understand that the spots that do not > qualify > > > the QC > > > > filter are given a weight of "0" by limma and > are > > > not > > > > considered for normalization and will not > affect > > > the > > > > analysis. > > > > The result file I get contains all the spots > > > (38000) > > > > in my case. > > > > > > Mmm, this doesn't make sense. You have 4 arrays > > > with 39000 spots on each array. Hence you have > > > 4*39000 spots, not 39000. The output from > > > topTable() gives a summarized log-ratio for each > > > probe, > > > not a result for each spot. > > > > > > Gordon > > > > > > > Didn't the spots that were bad get removed > from > > > the > > > > final result? > > > > If not what is the cut off value (B, p etc) > that I > > > > need to use to get a set of reliable spots(I > cant > > > use > > > > all the 38000) from my result file for my > > > analysis. > > > > Is there a fixed formula to derive the same as > the > > > > values vary with the analysis. > > > > Waiting for your reply, > > > > Thank you, > > > > -Ankit > > > > > > > > > > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > > >
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At 01:48 PM 17/05/2005, Ankit Pal wrote: >Dear Adai, >Thank you for the detailed response. >Correct me if I'm wrong. >What you are saying is that after applying the "fdr" >I give a cut off of 0.05 for the p-value and write all >those spots into an object (positives). >That means, I do not consider the B values. This is one way to proceed. Have you read the section in the User's Guide on "Statistics for differential expression" which discusses which statistic to use? >In essence, it is just a one sample t test afetr >adjusting the p-value and taking into consideration >all those spots that have a p-value < 0.05? > > From my code in a previous mail to Gordon Smyth, I >have done a quality control (QC) using parameters and >threshold values prescribed by genepix. >I know from experience, a large number of spots do not >get through the filter. >As I understand from LIMMA, a weight of "0" is given >to any spot that does not get through the QC filter. >How do I exclude these spots from the final result >summary file? These spots are already excluded. You seem to be confused by the fact that excluding a spot does not exclude the corresponding probe, since there are four spots for each probe. You will get a valid t-statistic and p-value for a probe if any one of the spots for that probe get a positive weight. (Note you can get a Bayesian t-statistic even for a probe with no residual df.) >I need to get a set of significantly differentially >expressed genes that have got through the QC filter. >How do I do it using LIMMA? This is what you have been getting all along. Gordon >Thank you, >-Ankit
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See comments below. If anyone finds them erroneous, please correct me. On Mon, 2005-05-16 at 20:48 -0700, Ankit Pal wrote: > Dear Adai, > Thank you for the detailed response. > Correct me if I'm wrong. > What you are saying is that after applying the "fdr" > I give a cut off of 0.05 for the p-value and write all > those spots into an object (positives). > That means, I do not consider the B values. In some sense, yes. In paragraph 2 of section 7 of [1], Symth says that posterior odds (B) depend on the estimate of the proportion of genes that are differentially expressed while moderated t-test (whose p-values are given in topTable output) does not. The B values are useful if you want to calculate posterior probabilities of differential expression. [1] Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1, Article 3. http://www.bepress.com/sagmb/vol3/iss1/art3/ > In essence, it is just a one sample t test afetr > adjusting the p-value and taking into consideration > all those spots that have a p-value < 0.05? No, this is based on moderated t-test where the variance in the denominator is shrunk towards a global variance like what SAM does. But unlike SAM, LIMMA has 1) a better theoretical derivation and shrinks the variance instead of standard deviation 2) shows that the moderated t-test follows a t-distribution with augmented degrees of freedom and thus no need to obtain p-values by permutation testing, hence computationally faster 3) more flexible to extend to other designs > >From my code in a previous mail to Gordon Smyth, I > have done a quality control (QC) using parameters and > threshold values prescribed by genepix. > I know from experience, a large number of spots do not > get through the filter. > As I understand from LIMMA, a weight of "0" is given > to any spot that does not get through the QC filter. > How do I exclude these spots from the final result > summary file? > I need to get a set of significantly differentially > expressed genes that have got through the QC filter. > How do I do it using LIMMA? I assume you have read the section 5.4 of the User Guide http://bioinf.wehi.edu.au/limma/usersguide.pdf I am not familiar with spot filtering nor genepix, so I cannot help you much here. But re-reading your previous mail, I noticed that you used something like "% > B635+2SD". Are you sure this is not "% > B635 + 2 SD". Note that the spaces matter ! Otherwise you can try renaming the columns to something like "green2sd" and "red2sd" just in case R is mis-reading the spaces somehow. Best wishes, Adai > Thank you, > -Ankit > > > > > --- Adaikalavan Ramasamy <ramasamy@cancer.org.uk> > wrote: > > > See comments below. > > > > On Mon, 2005-05-16 at 05:42 -0700, Ankit Pal wrote: > > > Dear Dr Smyth, > > > I guess there has been a mistake in communication. > > > There are 39000 spots on the array but the number > > of > > > probes are lesser as there are replicates(not a > > fixed > > > number) on the array. > > > The output from toptable() gives me a total of > > 39000 > > > rows, one for each spot. > > > I need to give an output of the spots most > > significant > > > in this list. > > > > I do not understand this clearly. Do you mean you > > have some duplicated > > spots or is there redundancy when you convert to > > something common like > > unigene ID ? > > > > If the former, I believe there are some facilities > > within LIMMA for > > equal number duplications per spots but I am not > > sure. For the latter, > > you could use tapply or something to find the > > min/max of the statistics. > > > > > What is the cut off value(p or B or any other) I > > need > > > to use above which I can consider a set of > > > differentially expressed genes significant enough > > for > > > further use (eg :function based clustering of all > > > significantly overexpressed genes). > > > I cannot use all the genes in the topTable() > > output. > > > Where do I put the cut off? > > > Is there a way I can calculate it? > > > > Selection of p-value threshold is arbitrary. However > > it would be a good > > idea to adjust for multiple comparison. My preferred > > method is the FDR > > by Benjamini and Hochberg but there are many other > > methods too each with > > a different meaning. I will illustrate FDR using the > > slightly modified > > example from help(topTable) : > > > > set.seed(123) # for reproducibility only - do not > > use this in real life > > > > M <- matrix(rnorm(10000*6,sd=0.3), 10000, 6) > > M[1:100, 1:3] <- M[1:100, 1:3] + 2 > > design <- cbind( First3Arrays=c(1,1,1,0,0,0), > > Last3Arrays=c(0,0,0,1,1,1)) > > fit <- lmFit(M, design=design) > > fit <- eBayes(fit) > > > > tab <- topTable( fit, adjust.method="fdr", > > sort.by="p", n=nrow(M) ) > > > > positives <- rownames(tab)[ which(tab[ , "P.Value"] > > < 0.05) ] > > > > The "positives" are the names of genes that we > > declare as differentially > > expression and it is expected that 5% of these genes > > are false positives > > on average. > > > > Note that in this case we can explicitly calculate > > the FP rate as > > follows because we have simulated the datasets. > > > > mean( ifelse( as.numeric(positives) <= 100, 0, 1 ) ) > > 0.06542056 > > > > I am sure Gordon Symth has a far more elegant and > > flexible version of > > this documented somewhere in his user guide. > > > > Regards, Adai > > > > > > > sincerely, > > > -Ankit > > > > > > --- Gordon K Smyth <smyth@wehi.edu.au> wrote: > > > > On Mon, May 16, 2005 9:49 pm, Ankit Pal said: > > > > > Dear Dr Smyth, > > > > > I'm sorry for not having specified which > > result > > > > file. > > > > > It is the final result summary we get after we > > > > give > > > > > the command > > > > > Resultfile <- topTable(fit,n=200, > > adjust="fdr") > > > > > A sample result file has been attached. > > > > > The code I used for my analysis is > > > > > > > > > >> targets <- readTargets("target.txt") > > > > > > > > > > #The QC filter > > > > >> myfun <- function(x,threshold=55){ > > > > > + okred <- abs(x[,"% > B635+2SD"]) > threshold > > > > > + okgreen <- abs(x[,"% > B532+2SD"]) > > > threshold > > > > > + okflag <- abs(x[,"Flags"]) > 0 > > > > > + okRGN <- abs(x[,"Rgn R"]) > 0.6 > > > > > + as.numeric(okgreen || okred || okflag || > > okRGN) > > > > > + } > > > > > #end of QC filter > > > > > > > > > >> RG_7 <- read.maimages(targets$FileName, > > > > > source="genepix",wt.fun=myfun) > > > > >> RG_7$genes <- readGAL() > > > > > > > > As I said last week, this command is not needed > > with > > > > GenePix data. You should omit it. > > > > > > > > >> RG_7$printer <- getLayout(RG_7$genes) > > > > >> MA_7 <- > > > > normalizeWithinArrays(RG_7,method="loess") > > > > >> MA_7 <- normalizeBetweenArrays(MA_7) > > > > >> fit_7 <- lmFit(MA_7, design=c(1,-1,1,-1)) > > > > >> fit_7 <- eBayes(fit_7) > > > > >> options(digits=3) > > > > >> Resultfile_7 <- topTable(fit_7, n=39000, > > > > > adjust="fdr") > > > > >> Resdat_7 <-data.frame(Resultfile_7) > > > > > > > > Resultfile_7 is already a data.frame. > > > > > > > > >> write.table(Resdat_,file='Result.csv',quote = > > > > FALSE, > > > > > sep = "\t") > > > > > > > > > > I understand that the spots that do not > > qualify > > > > the QC > > > > > filter are given a weight of "0" by limma and > > are > > > > not > > > > > considered for normalization and will not > > affect > > > > the > > > > > analysis. > > > > > The result file I get contains all the spots > > > > (38000) > > > > > in my case. > > > > > > > > Mmm, this doesn't make sense. You have 4 arrays > > > > with 39000 spots on each array. Hence you have > > > > 4*39000 spots, not 39000. The output from > > > > topTable() gives a summarized log-ratio for each > > > > probe, > > > > not a result for each spot. > > > > > > > > Gordon > > > > > > > > > Didn't the spots that were bad get removed > > from > > > > the > > > > > final result? > > > > > If not what is the cut off value (B, p etc) > > that I > > > > > need to use to get a set of reliable spots(I > > cant > > > > use > > > > > all the 38000) from my result file for my > > > > analysis. > > > > > Is there a fixed formula to derive the same as > > the > > > > > values vary with the analysis. > > > > > Waiting for your reply, > > > > > Thank you, > > > > > -Ankit > > > > > > > > > > > > > > > > > > _______________________________________________ > > > Bioconductor mailing list > > > Bioconductor@stat.math.ethz.ch > > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > > > > > > > > > __________________________________________________ > Do You Yahoo!? > Tired of spam? Yahoo! Mail has the best spam protection around > http://mail.yahoo.com >
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Ankit Pal ▴ 230
@ankit-pal-1242
Last seen 10.2 years ago
Dear Dr Smyth, I would prefer using the p-value with a threshold value of 0.05. In the case of an experiment(I sent you the code) I did, the reult file contains p-values (after fdr) in range of 0.96 - 0.97. How did I get such values and where do I place a cut off in this case? Am I doing something wrong? In the user guide it says "If none of the raw p-value are less than 1/G, where G is the number of genes included in the fit, then all of the adjusted p-values will the equal to 1.Since 1/G is about the expected size of the smallest p-values given purely random variation and uniform p-values, this means that there is no overall evidence of differential expression." I am not too sure I understand what it means. Should there be atleast one p-value < 1/G? I am attaching a sample data output file to this mail. What should be done in my case? Thank you, sincerely, -Ankit --- Gordon Smyth <smyth@wehi.edu.au> wrote: > At 01:48 PM 17/05/2005, Ankit Pal wrote: > >Dear Adai, > >Thank you for the detailed response. > >Correct me if I'm wrong. > >What you are saying is that after applying the > "fdr" > >I give a cut off of 0.05 for the p-value and write > all > >those spots into an object (positives). > >That means, I do not consider the B values. > > This is one way to proceed. Have you read the > section in the User's Guide > on "Statistics for differential expression" which > discusses which statistic > to use? > > >In essence, it is just a one sample t test afetr > >adjusting the p-value and taking into consideration > >all those spots that have a p-value < 0.05? > > > > From my code in a previous mail to Gordon Smyth, I > >have done a quality control (QC) using parameters > and > >threshold values prescribed by genepix. > >I know from experience, a large number of spots do > not > >get through the filter. > >As I understand from LIMMA, a weight of "0" is > given > >to any spot that does not get through the QC > filter. > >How do I exclude these spots from the final result > >summary file? > > These spots are already excluded. You seem to be > confused by the fact that > excluding a spot does not exclude the corresponding > probe, since there are > four spots for each probe. You will get a valid > t-statistic and p-value for > a probe if any one of the spots for that probe get a > positive weight. (Note > you can get a Bayesian t-statistic even for a probe > with no residual df.) > > >I need to get a set of significantly differentially > >expressed genes that have got through the QC > filter. > >How do I do it using LIMMA? > > This is what you have been getting all along. > > Gordon > > >Thank you, > >-Ankit > > __________________________________ -------------- next part -------------- Block Row Column ID Name M A t P.Value B 15254 19 26 17 NM_033613 scl0001747.1_14 1.4052 5.986 5.654 0.9627 -4.360 29822 37 26 23 AB117944 scl0003290.1_30 1.5988 5.571 5.557 0.9627 -4.362 18221 23 16 18 NM_033578 scl093703.1_214 1.3846 7.291 5.498 0.9627 -4.363 33734 42 21 25 NM_175677 scl00319236.1_170 1.2535 7.807 5.123 0.9627 -4.370 30787 39 2 18 - scl11230.1.1_125 1.5640 10.169 4.974 0.9627 -4.374 21676 27 24 21 NM_175657 scl00319161.1_8 1.3489 6.576 4.839 0.9627 -4.377 10983 14 18 7 NM_007999 scl014156.1_86 1.2005 4.078 4.821 0.9627 -4.377 26138 33 10 7 NM_028209 scl24036.11_204 1.4833 10.811 4.772 0.9627 -4.378 25635 32 21 16 NM_207298 scl0099151.1_201 1.2067 7.145 4.751 0.9627 -4.379 33449 42 11 10 - scl42410.1.3_0 -1.1950 6.997 -4.685 0.9627 -4.381 11683 15 14 6 - scl35668.26.1_1 1.3209 2.752 4.499 0.9627 -4.385 13694 17 28 21 - scl000055.1_1_REVCOMP 1.1755 6.008 4.496 0.9627 -4.386 14944 19 15 4 - scl27546.1.1069_39 -1.0644 6.182 -4.489 0.9627 -4.386 29762 37 24 17 NM_025284 scl0019240.2_329 1.2948 9.347 4.479 0.9627 -4.386 11789 15 18 4 XM_148353 scl067609.1_307 1.6845 3.191 4.433 0.9627 -4.387 2764 4 13 13 NM_178934 scl39088.5.1_30 1.1694 6.481 4.367 0.9627 -4.389 29678 37 21 14 NM_146075 scl00224640.2_275 1.2598 6.786 4.359 0.9627 -4.389 33435 42 10 23 - scl11006.1.1_298 -1.5154 4.933 -4.258 0.9627 -4.393 12481 16 13 22 NM_173731 scl36691.12_254 1.3582 2.918 4.248 0.9627 -4.393 19597 25 7 19 NM_147027 scl46618.1.187_149 2.2705 9.797 4.190 0.9627 -4.395 32290 40 28 10 NM_153798 scl0000113.1_1 1.0130 4.491 4.157 0.9627 -4.396 11762 15 17 4 - scl073362.2_30 1.4282 2.531 4.128 0.9627 -4.397 15866 20 19 9 - scl077449.2_20 1.3121 2.870 4.127 0.9627 -4.397 14762 19 8 11 NM_025536 scl47782.2_279 1.0271 5.729 4.109 0.9627 -4.397 27464 34 29 11 NC_001819 9629514_329 | Rauscher murine leukemia virus 1.2519 9.932 4.048 0.9627 -4.399 32482 41 5 14 NM_016875 scl41354.10.1_7 1.3216 10.076 4.030 0.9627 -4.400 9365 12 18 7 - scl069350.1_47 1.2922 4.009 4.003 0.9627 -4.401 15841 20 18 11 NM_027268 scl069938.2_27 1.7431 2.031 4.000 0.9627 -4.401 7426 10 6 10 - scl28349.3_240 1.1551 7.934 3.981 0.9627 -4.402 33257 42 4 7 NM_028243 scl32376.11_593 0.9688 6.692 3.977 0.9627 -4.402 15479 20 4 27 NM_146551 scl5601.1.1_211 0.9451 4.314 3.962 0.9627 -4.402 5896 8 9 17 NM_028108 scl49079.8_50 -1.2436 6.015 -3.962 0.9627 -4.402 21375 27 13 17 NM_007830 scl16375.7_22 0.9112 6.972 3.909 0.9627 -4.404 21793 27 29 3 K00604 IGHV1S16|K00604|Ig_heavy_variable_1S16_234 0.9863 6.598 3.904 0.9627 -4.404 29994 38 3 7 NM_018827 scl33727.4.1_6 1.0797 7.621 3.891 0.9627 -4.405 13393 17 17 17 NM_019737 scl056386.2_4 1.2250 3.139 3.871 0.9627 -4.406 15345 19 29 27 - AFFX- BioB-M-at_62 0.9412 6.040 3.864 0.9627 -4.406 29758 37 24 13 NM_013655 scl0020315.1_134 0.9538 5.124 3.861 0.9627 -4.406 15716 20 13 21 NM_175528 scl29095.10_195 1.3016 2.642 3.847 0.9627 -4.406 10670 14 6 18 XM_134476 scl34486.9.1_200 1.7255 9.395 3.839 0.9627 -4.407
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At 02:58 PM 17/05/2005, Ankit Pal wrote: >Dear Dr Smyth, >I would prefer using the p-value with a threshold >value of 0.05. >In the case of an experiment(I sent you the code) I >did, the reult file contains p-values (after fdr) in >range of 0.96 - 0.97. Clear result. No selected genes. End of story. >How did I get such values and where do I place a cut >off in this case? >Am I doing something wrong? >In the user guide it says >"If none of the raw p-value are less than 1/G, >where G is the number of genes included in the fit, >then all of the adjusted p-values will the >equal to 1.Since 1/G is about the expected size of the >smallest p-values given purely random variation and >uniform p-values, this means that there >is no overall evidence of differential expression." > >I am not too sure I understand what it means. >Should there be atleast one p-value < 1/G? No there need not be. The above text which you have quoted already tells you so. What would be the meaning of statistical significance, if you were guaranteed to get a significant result in every single experiment that you did? >I am attaching a sample data output file to this mail. >What should be done in my case? Perhaps take a more sophisticated approach to background correction and spot filtering. Or else accept that you don't have statistical significance for this data. Gordon >Thank you, >sincerely, >-Ankit
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