[BioC] Illumina beadlevel expression data

[Matt Ritchie]

Dear Ina,

I'm sorry to say that I haven't seen values like this before, and the X and
Y coordinates don't look unusual.  I have 2 further suggestions...

1.  Try running readIllumina with useImages=FALSE to see the values BeadScan
calculated for each bead.  Are they also negative?  Note that these will be
background corrected, so Gb will be set to 0 in BLData.

2.  Upgrade to R 2.6.0 and beadarray 1.6.0 (the C code in readIllumina
changed recently).  Do you still see strange values?  In future emails, it
would be helpful to include the output from sessionInfo().

If this doesn't uncover anything, perhaps you can send me the data off list
so that I can take a closer look.

Best wishes,

Matt

On 15/11/07 15:10, "Ina Hoeschele" <inah at vt.edu> wrote:

> 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,imageManipula
>>> ti
>>> 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