Hi all, I have cel-files from Affymetrix U133a+b chips and I wanted to do some quality control - so I read in the cel-files with the affy-package and got an affybatch-object. Then I had a look at the RNA degradation plots. I know that Microarray suite performs 3'/5'-ratios as a quality check. My problem is: I've only got the cel-files - there is no other information from Microarray suite available. So my question is: is it possible to calculate 3'/5'-ratios out of the cel-file? Thank you in advance for your help. Many greetings Sylvia

It is quite easy to calculate these ratios, but you would not do so directly from the .cel file (MAS5 doesn't use raw data either). Instead, calculate expression values using rma, expresso, whatever. Then you can extract the corresponding values to calculate the ratios. The probes that are spiked in as molecular biology controls are BioB, BioC, BioD and CreX. There are two sets of probes for each of the controls, so you may have to do some digging to see if one or both are used for the ratio. However, a naive approach would be to simply calculate the ratios for each chip using each probe, doing something like this: Firstchip.BioCs <- exprs(eset)["AFFX-BioB-3_st",1] - exprs(eset)["AFFX-BioB-5_st",1] Firstchip.BioCa <- exprs(eset)["AFFX-BioB-3_at",1] - exprs(eset)["AFFX-BioB-5_at",1] Secondchip.BioCs <- exprs(eset)["AFFX-BioB-3_st",2] - exprs(eset)["AFFX-BioB-5_st",2] etc, etc. This will give you the log ratios. You might want to take the antilog if that is what MAS5 usually reports. You can get all the names of the control probes by geneNames(eset). They are all near the bottom of the list. Here I am using eset as the name of the exprSet you get after calculating expression values. HTH, Jim James W. MacDonald Affymetrix and cDNA Microarray Core University of Michigan Cancer Center 1500 E. Medical Center Drive 7410 CCGC Ann Arbor MI 48109 734-647-5623 >>> Sylvia Merk <merk@ibe.med.uni-muenchen.de> 10/28/03 09:09AM >>> Hi all, I have cel-files from Affymetrix U133a+b chips and I wanted to do some quality control - so I read in the cel-files with the affy-package and got an affybatch-object. Then I had a look at the RNA degradation plots. I know that Microarray suite performs 3'/5'-ratios as a quality check. My problem is: I've only got the cel-files - there is no other information from Microarray suite available. So my question is: is it possible to calculate 3'/5'-ratios out of the cel-file? Thank you in advance for your help. Many greetings Sylvia _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor

Sorry - a typo - a 3 crept in instead of a 5 - it should be this.... qc.ratios <-function(x,logged=T) { x <- exprs(x); if(!logged) { x <- log2(x); } rats <- 2^cbind((x["AFFX-HUMGAPDH/M33197_3_at",] - x["AFFX-HUMGAPDH/M33197_5_at",]), (x["AFFX-HUMGAPDH/M33197_3_at",] - x["AFFX-HUMGAPDH/M33197_M_at",]), (x["AFFX-HSAC07/X00351_3_at",] - x["AFFX-HSAC07/X00351_5_at",]), (x["AFFX-HSAC07/X00351_3_at",] - x["AFFX-HSAC07/X00351_M_at",])); colnames(rats) <- c("GAPDH.3'/5'","GAPDH.3'/M","beta.actin.3'/5'","beta.actin.3'/M"); return(rats); } > -----Original Message----- > From: Crispin Miller > Sent: 28 October 2003 15:39 > To: 'Sylvia Merk' > Cc: Bioconductor (bioconductor@stat.math.ethz.ch) > Subject: RE: [BioC] 3' 5' ratio > > > Hi, > try this: > > qc.ratios <-function(x,logged=T) { > x <- exprs(x); > if(!logged) { x <- log2(x); } > rats <- 2^cbind((x["AFFX-HUMGAPDH/M33197_3_at",] - > x["AFFX-HUMGAPDH/M33197_5_at",]), > (x["AFFX-HUMGAPDH/M33197_3_at",] - > x["AFFX-HUMGAPDH/M33197_M_at",]), > (x["AFFX-HSAC07/X00351_3_at",] - > x["AFFX-HSAC07/X00351_3_at",]), > (x["AFFX-HSAC07/X00351_3_at",] - > x["AFFX-HSAC07/X00351_M_at",])); > colnames(rats) <- > c("GAPDH.3'/5'","GAPDH.3'/M","beta.actin.3'/5'","beta.actin.3'/M"); > return(rats); > } > > Where x is an AffyBatch object and the data has been > normalised using the mas5 algorithm... And logged says > whether the expression levels are log2() or not... > > It gives you 3'/5' ratios and also 3'/mid ratios for GAPDH > and beta-actin... It should work - I've checked it for one > (hgu133a) array ;-) > > Cheers, > Crispin > > > > > -----Original Message----- > > From: Sylvia Merk [mailto:merk@ibe.med.uni-muenchen.de] > > Sent: 28 October 2003 14:10 > > To: bioconductor@stat.math.ethz.ch > > Subject: [BioC] 3' 5' ratio > > > > > > Hi all, > > > > I have cel-files from Affymetrix U133a+b chips and I wanted > > to do some > > quality control - so I read in the cel-files with the > > affy-package and > > got an affybatch-object. > > Then I had a look at the RNA degradation plots. I know that > > Microarray > > suite performs 3'/5'-ratios as a quality check. My problem > > is: I've only > > got the cel-files - there is no other information from > > Microarray suite > > available. So my question is: is it possible to calculate > > 3'/5'-ratios > > out of the cel-file? > > Thank you in advance for your help. > > > > Many greetings > > Sylvia > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://www.stat.math.ethz.ch/mailman/listinfo> /bioconductor > > > -------------------------------------------------------- This email is confidential and intended solely for the use o...{{dropped}}

Hi, try this: qc.ratios <-function(x,logged=T) { x <- exprs(x); if(!logged) { x <- log2(x); } rats <- 2^cbind((x["AFFX-HUMGAPDH/M33197_3_at",] - x["AFFX-HUMGAPDH/M33197_5_at",]), (x["AFFX-HUMGAPDH/M33197_3_at",] - x["AFFX-HUMGAPDH/M33197_M_at",]), (x["AFFX-HSAC07/X00351_3_at",] - x["AFFX-HSAC07/X00351_3_at",]), (x["AFFX-HSAC07/X00351_3_at",] - x["AFFX-HSAC07/X00351_M_at",])); colnames(rats) <- c("GAPDH.3'/5'","GAPDH.3'/M","beta.actin.3'/5'","beta.actin.3'/M"); return(rats); } Where x is an AffyBatch object and the data has been normalised using the mas5 algorithm... And logged says whether the expression levels are log2() or not... It gives you 3'/5' ratios and also 3'/mid ratios for GAPDH and beta-actin... It should work - I've checked it for one (hgu133a) array ;-) Cheers, Crispin > -----Original Message----- > From: Sylvia Merk [mailto:merk@ibe.med.uni-muenchen.de] > Sent: 28 October 2003 14:10 > To: bioconductor@stat.math.ethz.ch > Subject: [BioC] 3' 5' ratio > > > Hi all, > > I have cel-files from Affymetrix U133a+b chips and I wanted > to do some > quality control - so I read in the cel-files with the > affy-package and > got an affybatch-object. > Then I had a look at the RNA degradation plots. I know that > Microarray > suite performs 3'/5'-ratios as a quality check. My problem > is: I've only > got the cel-files - there is no other information from > Microarray suite > available. So my question is: is it possible to calculate > 3'/5'-ratios > out of the cel-file? > Thank you in advance for your help. > > Many greetings > Sylvia > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://www.stat.math.ethz.ch/mailman/listinfo> /bioconductor > -------------------------------------------------------- This email is confidential and intended solely for the use o...{{dropped}}