Hi Arnar:

	The number of negative control probes does not seem to match that in HT12 v3. Maybe you can check the difference in negative control probe sequences between these two versions. You can find the sequence information from Illumina's manifest files. I had been looking at the intensity distribution of negative controls in hundreds of BeadChip arrays (but not including the new HT12 v4) and I have never seen the distributions as shown in your figures. One possibility is that Illumina changed their negative control probe sequences and the new controls are not as good as the old controls. But it is also possible that what you saw only occurred to your data. 

	Hope this helps.

Wei

On Feb 23, 2011, at 7:38 PM, Arnar Flatberg wrote:

> Hi Wei,
> 
> On Tue, Feb 22, 2011 at 11:14 PM, Wei Shi <shi@wehi.edu.au> wrote:
> Dear Arnar:
> 
>        Thanks for the detailed report for the intensity distributions of negative control probes in your data. But could you please provide more information about the numbers of different types of probes?
> 
> I'm not quite sue what you are referring to here. I'll try to explain in more detail, and I'll provide some code for reproduction at the bottom.
> 
> I'm only considering the control-probes on the Illumina chip, that is the 770 negative (If I'm not mistaken, I believe these are non-human permuted sequences). I dont use the rest of the controls, that is, the hybridization- , labeliing-, biotin-, housekeeping-, stringency-, and the brand new spike-in-controls.
> 
> I'm investigating these as representative of my background signal.  
> 
>  
> If you used the limma function read.ilmn to read in your data, you can get this information by this command:
> 
> table(x$genes$Status)
> 
>        Also, it will be a lot helpful if you could provide your code for creating the density plots.
> 
> I'm using the `lumi` package for reading and plotting data, below is code for reproduction
> 
> require(lumi)
> controlDataFileName <- "filename_of_beadstudio_control_data_export"
> C1 <- getControlData(controlDataFileName)
> n <- dim(C1)[1]
> neg.probes <-  as.matrix(C1[C1[,1]=="NEGATIVE",3:n])
> x11(); plotDensity(neg.probes, main="HT12-v4 Example", addLegend=FALSE)
> 
> 
> > sessionInfo()
> R version 2.12.1 (2010-12-16)
> Platform: x86_64-pc-linux-gnu (64-bit)
> 
> locale:
>  [1] LC_CTYPE=en_US.utf8       LC_NUMERIC=C             
>  [3] LC_TIME=en_US.utf8        LC_COLLATE=en_US.utf8    
>  [5] LC_MONETARY=C             LC_MESSAGES=en_US.utf8   
>  [7] LC_PAPER=en_US.utf8       LC_NAME=C                
>  [9] LC_ADDRESS=C              LC_TELEPHONE=C           
> [11] LC_MEASUREMENT=en_US.utf8 LC_IDENTIFICATION=C      
> 
> attached base packages:
> [1] stats     graphics  grDevices utils     datasets  methods   base     
> 
> other attached packages:
> [1] lumi_2.2.0     Biobase_2.10.0
> 
> loaded via a namespace (and not attached):
>  [1] affy_1.28.0           affyio_1.18.0         annotate_1.28.0      
>  [4] AnnotationDbi_1.12.0  DBI_0.2-5             grid_2.12.1          
>  [7] hdrcde_2.14           KernSmooth_2.23-4     lattice_0.19-13      
> [10] MASS_7.3-7            Matrix_0.999375-46    methylumi_1.4.0      
> [13] mgcv_1.7-2            nlme_3.1-97           preprocessCore_1.12.0
> [16] RSQLite_0.9-2         xtable_1.5-6       
> 
> 
> 
> Thanks, 
> 
> 
> Arnar
> 
> 


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