[BioC] NAs introduced by marrayNorm after background subtraction with Limma

[Ellen Sebastian [guest]]

Hello,

I am trying to use Limma for background subtraction and then marray for normalization. I am confused about why normalization is introducing NA values.

This happens regardless of which background subtraction and normalization methods I use - even edwards, which does not produce ANY negative fluorescence values after background subtraction. The only way to avoid SOME (not all) of the NAs is to not use background subtraction at all.

Where are these NAs coming from, and how can I avoid them? Why is using edwards background subtraction (which produces no negative fluorescence values) still introducing NAs?

The only legitimate reason I can think of for normalization to produce NAs is if there were negative fluorescence values - but these NAs are present even if there are no negative fluorescence values. For example, probe 7 has green = 107, red = 159, normalized log2 ratio = NA.
(I doubt you're going to be able to read this data matrix but here it is anyway. Basically it says that NAs are introduced even if there are no negative fluorescence values.)
 
reporter	Green BG subtracted, "edwards"	red BG subtracted," edwards"	BG subtracted "edwards", normalized loess	Green BG subtracted, "subtract"	red BG subtracted, "subtract"	BG subtracted "subtract", normalized loess	Green, no BG subtraction	Red, no BG subtraction	 no BG subtraction, normalized loess
1	1161 	1238	     0.135468422	1161	1238	0.143848955	1280	1303	0.094990556
7	107	159	NA	107	159	NA	224	224	0.352352478
15	8.881238564	51	NA	-4	51	NA	118	118	NA
18	49	90	NA	49	90	NA	175	157	0.761815105
21	7.770487011	44	NA	-18	44	NA	111	113	NA


another question: I am using the following code to splice in my background-subtraced data from Limma into an existant marrayRaw object:

RGraws<-BGsubbed<-read.maimages(files[i],"genepix",wt.fun=wtflags(0.1), verbose=FALSE)
      RGsubbed<-backgroundCorrect(RGraw, method = BGsubmethod, verbose = FALSE)
      BGsubbedGPR<-GPRs[[i]]
      # splice in background-subtracted data into marray object
      BGsubbed at maRf<-RGsubbed$R
      BGsubbed at maGf<-RGsubbed$G

However, this code leaves the maRb and maGb slots in BGsubbed intact. Does this cause Marray to perform background subtraction again, even though I have already done background subtraction in Limma? should I be overwriting BGsubbed at maGb and BGsubbed at maRb with 0's?

Thanks for any help. I have been banging my head against the wall for days trying to figure out where these NAs come from.

 -- output of sessionInfo(): 

R version 2.15.2 (2012-10-26)
Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit)

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

other attached packages:
[1] marray_1.36.0 limma_3.14.4 

loaded via a namespace (and not attached):
[1] csSAM_1.2.1  tools_2.15.2

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