Hello all, I have a set of 'boutique' Agilent arrays (ie. they are functional gene arrays for which either a large or small number of gene can be differenitally expressed). We have printed a few different sets of control spots to use for within array normalization. I have pasted by script below, and the error message and warnings. Because of the warnings I thought that the set of control spots (6 out of 3600 total) might be too small to fit a loess curve through so I then tried with another 'control set" for which there are 220 spots . And I still recieved the error " Error in qr.default(x) : NA/NaN/Inf in foreign function call (arg 1) ", but there were no additional warnings about too few degrees of freedom. Any thoughts on why I'm recieving this error message? Thanks, Alison Partial Script >>>>>> library(limma) targets<-readTargets("EPBDIExprTargets.txt") RG<-read.maimages(targets$FileName,source='genepix',wt.fun=wtflags(0.1 )) spottypes<-readSpotTypes("SpotTypesEP.txt") RGnm<-backgroundCorrect(RG,method='normexp') RGnm$genes$Status<-controlStatus(spottypes,RG) ControlSpots<-grep("Control",RGnm$genes$Status) MAnmcntrl<- normalizeWithinArrays(RGnm,method='control',controlspots=ControlSpots) MAnmcntrlAqu<-normalizeBetweenArrays(MAnmcntrl,method='Aquantile') Partial Output >>>>>>>>>>>>>> Corrected array 9 Red channel Corrected array 1 Corrected array 2 Corrected array 3 Corrected array 4 Corrected array 5 Corrected array 6 Corrected array 7 Corrected array 8 Corrected array 9 Matching patterns for: ID Found 3600 All Found 6 Control Setting attributes: values col cex Error in qr.default(x) : NA/NaN/Inf in foreign function call (arg 1) In addition: There were 50 or more warnings (use warnings() to see the first 50) > warnings() Warning messages: 1: span too small. fewer data values than degrees of freedom. 2: zero-width neighborhood. make span bigger 3: zero-width neighborhood. make span bigger 4: zero-width neighborhood. make span bigger 5: zero-width neighborhood. make span bigger 6: zero-width neighborhood. make span bigger 7: zero-width neighborhood. make span bigger 8: Chernobyl! trL<k 0="" 9:="" chernobyl!="" trl<k="" 0="" 10:="" span="" too="" small.="" fewer="" data="" values="" than="" degrees="" of="" freedom.="" 11:="" zero-width="" neighborhood.="" make="" span="" bigger="" 12:="" zero-width="" neighborhood.="" make="" span="" bigger="" 13:="" zero-width="" neighborhood.="" make="" span="" bigger="" 14:="" zero-width="" neighborhood.="" make="" span="" bigger="" 15:="" zero-width="" neighborhood.="" make="" span="" bigger="" ---------------------------------------------------------="" alison="" waller="" ph.d="" alison.waller="" at="" utoronto.ca="" <="" div="">

Hi Alison, Without knowing the biological details of your experiment, I agree that modifyWeights or just plain lowess normalization looks appropriate. These are the approaches that I generally recommend. BTW, it would appear that your email was blocked from the Bioconductor mailing list because of the large attachments. Best wishes Gordon On Wed, 22 Jul 2009, Alison Waller wrote: > Thank you for the reply Gordon, > > Yes, I should have looked at the plots earlier. It appears as if the second > set (16S) is not appropriate (due to variation in their response) and the > first set (Arab) is too small as you mentioned. > > It also appears as if the majority of the spots are not differentially > expressed so I can probably just use loess normalization on all of the spots. > > However, I don't want to totally ignore the data from the control spots, as > for some arrays the Arab control spots are further from M=0 than others, see > MA plots below. > > I decided to try using the modifyWeights function. > > The MA plots resulting from this look reasonable. Do you think this is a > valid approach given the small number of control spots, and the fact that > they have higher intensities than the majority of the spots? > > w<-modifyWeights(array(1,dim(RGnm)), RGnm$genes$Status, c("All","Arab"), > c(1,2)) > MAnmw<-normalizeWithinArrays(RGnm,weights=w) > > >