Hi All, I have just started using R / Bioconductor within the last couple of weeks to analyze our microarray data. While attempting to do the Print Tip Lowess normalization on a particular scan, I received the following error, and was unable to proceed with the analysis. I did a google search for the error message, but I am still unclear about what is wrong with my data. I have provided the warning I recieved, as well as the list of warning that I requested. If anyone can provide some suggestions, I will be extremely grateful. There are a fairly large number of NA's in my data. I am using R 2.0 with the most recent version of Bioconductor. Analysis is being done on a Gateway PC. Thanks in advance, Tony > Error in simpleLoess(y, x, w, span, degree, parametric, drop.square, > normalize, : > 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: zero-width neighborhood. make span bigger > 2: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 3: zero-width neighborhood. make span bigger > 4: zero-width neighborhood. make span bigger > 5: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 6: zero-width neighborhood. make span bigger > 7: zero-width neighborhood. make span bigger > 8: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 9: zero-width neighborhood. make span bigger > 10: zero-width neighborhood. make span bigger > 11: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 12: zero-width neighborhood. make span bigger > 13: zero-width neighborhood. make span bigger > 14: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 15: zero-width neighborhood. make span bigger > 16: zero-width neighborhood. make span bigger > 17: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 18: zero-width neighborhood. make span bigger > 19: zero-width neighborhood. make span bigger > 20: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 21: zero-width neighborhood. make span bigger > 22: zero-width neighborhood. make span bigger > 23: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 24: zero-width neighborhood. make span bigger > 25: zero-width neighborhood. make span bigger > 26: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 27: zero-width neighborhood. make span bigger > 28: zero-width neighborhood. make span bigger > 29: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 30: zero-width neighborhood. make span bigger > 31: zero-width neighborhood. make span bigger > 32: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 33: zero-width neighborhood. make span bigger > 34: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 35: zero-width neighborhood. make span bigger > 36: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 37: zero-width neighborhood. make span bigger > 38: zero-width neighborhood. make span bigger > 39: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 40: zero-width neighborhood. make span bigger > 41: zero-width neighborhood. make span bigger > 42: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 43: zero-width neighborhood. make span bigger > 44: zero-width neighborhood. make span bigger > 45: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 46: zero-width neighborhood. make span bigger > 47: zero-width neighborhood. make span bigger > 48: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 49: zero-width neighborhood. make span bigger > 50: zero-width neighborhood. make span bigger Tony Kowal Graduate Student Department of Cell and Molecular Biology Northwestern University Chisholm Lab 303 E. Chicago Ave. Rm 11-100 Chicago, IL 60611 e-mail: askowal@northwestern.edu

Quite simply, your data contains NA values. These can occur if your background subtracted values are <= 0 in one of the channels, leading to a nonsensical log(ratio) value. If you have a lot of NA values, this can be a problem, especially for print tip loess, as each print tip group has less values to work with, if lots of them are NA values then the loess fit may not be representative of your data. I have seen a log(ratio) switch from -5 to +5 due to this problem. To reduce the number of NA values you have, try one of the following: do not subtract background; use kooperberg background correction; subtract background and then set all values below a threshold to that threshold. Mick -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces@stat.math.ethz.ch] On Behalf Of Tony Kowal Sent: 06 December 2004 02:56 To: bioconductor@stat.math.ethz.ch Subject: [BioC] A question about lowess normalization Hi All, I have just started using R / Bioconductor within the last couple of weeks to analyze our microarray data. While attempting to do the Print Tip Lowess normalization on a particular scan, I received the following error, and was unable to proceed with the analysis. I did a google search for the error message, but I am still unclear about what is wrong with my data. I have provided the warning I recieved, as well as the list of warning that I requested. If anyone can provide some suggestions, I will be extremely grateful. There are a fairly large number of NA's in my data. I am using R 2.0 with the most recent version of Bioconductor. Analysis is being done on a Gateway PC. Thanks in advance, Tony > Error in simpleLoess(y, x, w, span, degree, parametric, drop.square, > normalize, : > 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: zero-width neighborhood. make span bigger > 2: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 3: zero-width neighborhood. make span bigger > 4: zero-width neighborhood. make span bigger > 5: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 6: zero-width neighborhood. make span bigger > 7: zero-width neighborhood. make span bigger > 8: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 9: zero-width neighborhood. make span bigger > 10: zero-width neighborhood. make span bigger > 11: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 12: zero-width neighborhood. make span bigger > 13: zero-width neighborhood. make span bigger > 14: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 15: zero-width neighborhood. make span bigger > 16: zero-width neighborhood. make span bigger > 17: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 18: zero-width neighborhood. make span bigger > 19: zero-width neighborhood. make span bigger > 20: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 21: zero-width neighborhood. make span bigger > 22: zero-width neighborhood. make span bigger > 23: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 24: zero-width neighborhood. make span bigger > 25: zero-width neighborhood. make span bigger > 26: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 27: zero-width neighborhood. make span bigger > 28: zero-width neighborhood. make span bigger > 29: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 30: zero-width neighborhood. make span bigger > 31: zero-width neighborhood. make span bigger > 32: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 33: zero-width neighborhood. make span bigger > 34: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 35: zero-width neighborhood. make span bigger > 36: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 37: zero-width neighborhood. make span bigger > 38: zero-width neighborhood. make span bigger > 39: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 40: zero-width neighborhood. make span bigger > 41: zero-width neighborhood. make span bigger > 42: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 43: zero-width neighborhood. make span bigger > 44: zero-width neighborhood. make span bigger > 45: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 46: zero-width neighborhood. make span bigger > 47: zero-width neighborhood. make span bigger > 48: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > 49: zero-width neighborhood. make span bigger > 50: zero-width neighborhood. make span bigger Tony Kowal Graduate Student Department of Cell and Molecular Biology Northwestern University Chisholm Lab 303 E. Chicago Ave. Rm 11-100 Chicago, IL 60611 e-mail: askowal@northwestern.edu _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioconductor

There is another way to overcome this type of problem, that uses a smooth background correction to spots that have intensity < background. This method was proposed by David Edwards in 2003 and was published in Bioinformatics, v 19, pp 825-833. Best regards, Gustavo On Sun, 5 Dec 2004 20:56:15 -0600, Tony Kowal <askowal@northwestern.edu> wrote: > Hi All, > > I have just started using R / Bioconductor within the last couple of > weeks to analyze our microarray data. While attempting to do the Print > Tip Lowess normalization on a particular scan, I received the following > error, and was unable to proceed with the analysis. I did a google > search for the error message, but I am still unclear about what is > wrong with my data. I have provided the warning I recieved, as well as > the list of warning that I requested. If anyone can provide some > suggestions, I will be extremely grateful. > > There are a fairly large number of NA's in my data. I am using R 2.0 > with the most recent version of Bioconductor. Analysis is being done > on a Gateway PC. > > Thanks in advance, > Tony > > > Error in simpleLoess(y, x, w, span, degree, parametric, drop.square, > > normalize, : > > 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: zero-width neighborhood. make span bigger > > 2: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 3: zero-width neighborhood. make span bigger > > 4: zero-width neighborhood. make span bigger > > 5: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 6: zero-width neighborhood. make span bigger > > 7: zero-width neighborhood. make span bigger > > 8: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 9: zero-width neighborhood. make span bigger > > 10: zero-width neighborhood. make span bigger > > 11: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 12: zero-width neighborhood. make span bigger > > 13: zero-width neighborhood. make span bigger > > 14: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 15: zero-width neighborhood. make span bigger > > 16: zero-width neighborhood. make span bigger > > 17: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 18: zero-width neighborhood. make span bigger > > 19: zero-width neighborhood. make span bigger > > 20: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 21: zero-width neighborhood. make span bigger > > 22: zero-width neighborhood. make span bigger > > 23: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 24: zero-width neighborhood. make span bigger > > 25: zero-width neighborhood. make span bigger > > 26: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 27: zero-width neighborhood. make span bigger > > 28: zero-width neighborhood. make span bigger > > 29: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 30: zero-width neighborhood. make span bigger > > 31: zero-width neighborhood. make span bigger > > 32: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 33: zero-width neighborhood. make span bigger > > 34: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 35: zero-width neighborhood. make span bigger > > 36: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 37: zero-width neighborhood. make span bigger > > 38: zero-width neighborhood. make span bigger > > 39: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 40: zero-width neighborhood. make span bigger > > 41: zero-width neighborhood. make span bigger > > 42: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 43: zero-width neighborhood. make span bigger > > 44: zero-width neighborhood. make span bigger > > 45: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 46: zero-width neighborhood. make span bigger > > 47: zero-width neighborhood. make span bigger > > 48: ihat=1 (diag L) in l2fit only makes sense if z=x (eval=data). > > 49: zero-width neighborhood. make span bigger > > 50: zero-width neighborhood. make span bigger > > Tony Kowal > Graduate Student > Department of Cell and Molecular Biology > Northwestern University > Chisholm Lab > 303 E. Chicago Ave. > Rm 11-100 > Chicago, IL 60611 > > e-mail: askowal@northwestern.edu > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > -- _______________________________________________________________ "The truth you speak has no past and no future. It is, and that's all it needs to be." "A verdade que voc? fala n?o tem passado nem futuro. Ela ?, e isso ? tudo que ela precisa ser." Autor desconhecido _______________________________________________________________ Gustavo Henrique Esteves e-mail: gesteves@gmail.com Home Page: http://www.vision.ime.usp.br/~gesteves/

> Date: Mon, 6 Dec 2004 07:52:44 -0200 > From: Gustavo Henrique Esteves <gesteves@gmail.com> > Subject: Re: [BioC] A question about lowess normalization > To: Tony Kowal <askowal@northwestern.edu> > Cc: bioconductor@stat.math.ethz.ch > Message-ID: <a79825cf041206015276d69a51@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > There is another way to overcome this type of problem, that uses a > smooth background correction to spots that have intensity < > background. This method was proposed by David Edwards in 2003 and was > published in Bioinformatics, v 19, pp 825-833. This and other background correction methods are available in the limma package. Gordon > Best regards, > Gustavo > > On Sun, 5 Dec 2004 20:56:15 -0600, Tony Kowal <askowal@northwestern.edu> wrote: >> Hi All, >> >> I have just started using R / Bioconductor within the last couple of >> weeks to analyze our microarray data. While attempting to do the Print >> Tip Lowess normalization on a particular scan, I received the following >> error, and was unable to proceed with the analysis. I did a google >> search for the error message, but I am still unclear about what is >> wrong with my data. I have provided the warning I recieved, as well as >> the list of warning that I requested. If anyone can provide some >> suggestions, I will be extremely grateful. >> >> There are a fairly large number of NA's in my data. I am using R 2.0 >> with the most recent version of Bioconductor. Analysis is being done >> on a Gateway PC. >> >> Thanks in advance, >> Tony >> >> > Error in simpleLoess(y, x, w, span, degree, parametric, drop.square, >> > normalize, : [listing of 50 warnings deleted] >> >> Tony Kowal >> Graduate Student >> Department of Cell and Molecular Biology >> Northwestern University >> Chisholm Lab >> 303 E. Chicago Ave. >> Rm 11-100 >> Chicago, IL 60611 >> >> e-mail: askowal@northwestern.edu