Quoting James Morris <slimjimbob1 at="" hotmail.com="">:
> Hello everyone,
>
> I am trying to use the MANOR spatial normalisation package but I
> have got a problem
>
> I get this
> message when i calculate the log2 ratios
>
> Warning message:
> NaNs produced in: log(x, base)
>
> and when I try to normalise I get
>
> Warning message:
> NaNs produced in: log(x, base)
>
> any help would be most appreciated
>
> James
>
> the script I am using:
[...]
Hi James,
I think the culprit is here:
## compute a log-ratio
ac$arrayValues$Ratio<-(ac$arrayValues$F635.Mean-
ac$arrayValues$B635.Mean)/(ac$arrayValues$F532.Mean-
ac$arrayValues$B532.Mean)
ac$arrayValues$Ratio[ac$arrayValues$Ratio==Inf] <- NA
ac$arrayValues$LogRatio <- log(ac$arrayValues$Ratio,2)
It's not unusual to have local background estimates that are larger
than the actual signal, so you get a negative signal after background
substraction... and the error you get is produced when trying to
calculate the log of a negative number.
You will have to either ignore the warnings (but you will lose the
spots that produce the error), or use a different background estimate,
or use a different background correction method, or even not do
background correction at all (if the slides are decent, no background
correction is often better than background substraction).
I use the 'limma' package, which offers a nice range of methods for
both background correction and normalisation. In particular, the
'normexp' background correction method seems to work very well in my
hands, when you use the correct offset. You could use limma for the
background correction, and then continue as you are doing... I am not
familiar with the Manor package, but I'd be surprised if you cannot
find in limma a method to your liking.
regards,
Jose
--
Dr. Jose I. de las Heras Email: J.delasHeras at
ed.ac.uk
The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131
6513374
Institute for Cell & Molecular Biology Fax: +44 (0)131
6507360
Swann Building, Mayfield Road
University of Edinburgh
Edinburgh EH9 3JR
UK
