[BioC] Illumina beadlevel expression data

[Ina Hoeschele]

Thank you, Matt.

Matt Ritchie wrote:
> Hi Ina,
>
> What version of beadarray are you using?  In older versions funny background
> values have been reported on some operating systems - this should be fixed
> on the release version though (beadarray 1.6.0 available on R-2.6.0).
>   
I am using R-2.5.1 (not sure what version of beadarray), which is still 
the current released version. Do I have to go to R-2.6.0?
> The first beads in BLData are typically ones which could not be decoded
> properly (ProbeIDs 0).  If these beads are too close to the edge of an
> image, or off the image, then zero foreground or background intensities may
> occur.  Check the X (GrnX) and Y (GrnY) coordinates for these beads - any
> with negative values are off the image, and cannot be quantified.
>
> BLData[[1]][1:10,]
>   
I have a total of 319952 beads. So I printed the information for the 
first 10 and for 10 others towards the end. Even for the first 10 beads 
the GrnX and GrnY are not negative. Here it is.
>  BLData[[1]][1:10,]
   ProbeID          G Gb      GrnX      GrnY
1    10008  0.0000000  0 1556.7592 13526.200
2    10008  2.5249867  0 1713.2862 17711.700
3    10008 -0.0359710  0   45.9317  6676.658
4    10008  0.0000000  0 1401.8142  2153.181
5    10008  0.0000000  0 2078.2252 15974.100
6    10008 -6.8111267  0 1193.8842  8654.250
7    10008  0.0000000  0   45.0026 13990.970
8    10008 77.2345222  0  542.1487 14834.780
9    10008 -0.8237067  0 1341.1462 13791.170
10   10008 -0.2324444  0 1312.7072  7549.003
>
>  BLData[[1]][300000:300010,]
       ProbeID           G Gb      GrnX      GrnY
300000 2140131  0.00000000  0 1258.9752 17301.580
300001 2140131 -0.24859133  0  398.1137  6108.199
300002 2140131 -0.07388444  0 2094.6222 16298.300
300003 2140131  0.00000000  0  642.9227  2094.740
300004 2140131 11.67171933  0 1352.2322  6294.191
300005 2140131  0.00000000  0 1104.5312 14069.740
300006 2140131 -0.15372500  0  963.8952  9653.909
300007 2140131  0.00000000  0  161.5619 14317.910
300008 2140131 -0.08331556  0 1515.5492 13932.460
300009 2140131  0.61296444  0 1934.0542 15118.710
300010 2140131  0.00000000  0  177.0217 16717.080

> If this doesn't shed any light, perhaps you can put the .tif and .txt files
> from the strip online so that I can take a closer look.
>   
I'll do that next if you don't have any other suggestions.
Many thanks for your time.
Ina

> Best wishes,
>
> Matt
>
>   
>> Hi,
>>    I am new to the analysis of Illumina beadlevel expression data. I
>> have data from a collaborator and have started to work with the data on
>> the first Beadchip (with 12 arrays or strips).  These are single channel
>> data. I am using the beadarray package.  I read the data in as follows:
>> BLData <- 
>> readIllumina(textType=".txt",useImages=TRUE,singleChannel=TRUE,imageManipulati
>> on="sharpen",
>> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod="none",offset=0,
>> normalizeMethod="none")
>> Note: above I'm doing NO background correction and note that I use
>> backgroundSize=17.
>>
>> Then I looked at the data:
>>
>> an <- arrayNames(BLData)
>> BLData at beadData[[an[1]]]$G[1:10]
>>  [1]  0.0000000  2.5249867 -0.0359710  0.0000000  0.0000000 -6.8111267
>>  [7]  0.0000000 77.2345222 -0.8237067 -0.2324444
>> BLData at beadData[[an[1]]]$Gb[1:10]
>>  [1] 0 0 0 0 0 0 0 0 0 0
>>
>> so I get quite a few G intensities that are zero and ALL of my
>> background intensities are zero - why is that ?????
>>
>> Then I created BLData with backgroundSize=4 and backgroundSize=40 (not
>> knowing what value I should be using ?????). For backgroundSize=4, I
>> find that
>> BLData at beadData[[an[1]]]$Gb[1:100]
>>   [1] -1  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0
>> 0  0  0
>>  [26]  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  2  0
>> 0  0  0
>>  [51]  0  0 24  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0
>> 0  0  0
>>  [76]  0  0  0  0  0  0  0  0  0  0 19  0  0  0  0  0  0  0  0  0  0  0
>> 0  0  0
>> so now I still have many zero Gb values, but also some nonzero ones and
>> even negative (-1)???
>> But when I use backgroundSize=40, then R crashes (repeatedly).
>>
>> And when I create BLData with backgroundMethod="minimum" and
>> backgroundSize=17, then I find the following G intensities:
>> BLData at beadData[[an[1]]]$G[1:10]
>>  [1] 2.425286e-12 2.524987e+00 2.425286e-12 2.425286e-12 2.425286e-12
>> 2.425286e-12 2.425286e-12 7.723452e+01 2.425286e-12
>> [10] 2.425286e-12
>>
>> These G and Gb values all look suspect to me - can someone please help?
>>
>> Many thanks, Ina
>>
>> _______________________________________________
>> Bioconductor mailing list
>> Bioconductor at stat.math.ethz.ch
>> https://stat.ethz.ch/mailman/listinfo/bioconductor
>> Search the archives:
>> http://news.gmane.org/gmane.science.biology.informatics.conductor
>>