[BioC] Illumina beadlevel expression data
Matt Ritchie
Matt.Ritchie at cancer.org.uk
Fri Nov 16 11:55:07 CET 2007
Dear Ina,
I'd suggest that you use the values in the text files (useImages=FALSE) as
these look reasonable, whereas the ones obtained from redoing the image
analysis look strange. The values in the text file are already background
corrected (foreground - background).
As I mentioned in previous emails, if you send me the data off-list, I can
take a closer look to try and work out what is going on.
Best wishes,
Matt
On 15/11/07 23:39, "Ina Hoeschele" <inah at vt.edu> wrote:
> Matt Ritchie wrote:
>> 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.
>>
> those values are different but still look strange to me (were they
> computed using the subtract or minimum method for background
> correction?) - here they are:
>> BLData[[1]][1:10,]
> ProbeID G Gb GrnX GrnY
> 1 10008 56 0 1556.7592 13526.200
> 2 10008 46 0 1713.2862 17711.700
> 3 10008 4 0 45.9317 6676.658
> 4 10008 37 0 1401.8142 2153.181
> 5 10008 48 0 2078.2252 15974.100
> 6 10008 65 0 1193.8842 8654.250
> 7 10008 129 0 45.0026 13990.970
> 8 10008 63 0 542.1487 14834.780
> 9 10008 24 0 1341.1462 13791.170
> 10 10008 45 0 1312.7072 7549.003
>> BLData[[1]][300000:300010,]
> ProbeID G Gb GrnX GrnY
> 300000 2140131 4 0 1258.9752 17301.580
> 300001 2140131 4 0 398.1137 6108.199
> 300002 2140131 0 0 2094.6222 16298.300
> 300003 2140131 3 0 642.9227 2094.740
> 300004 2140131 0 0 1352.2322 6294.191
> 300005 2140131 -1 0 1104.5312 14069.740
> 300006 2140131 22 0 963.8952 9653.909
> 300007 2140131 2 0 161.5619 14317.910
> 300008 2140131 0 0 1515.5492 13932.460
> 300009 2140131 0 0 1934.0542 15118.710
> 300010 2140131 -1 0 177.0217 16717.080
>>
>
>> 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().
>>
> I did upgrade to R-2.6.0 and beadarray 1.6.0 and I am getting the exact
> same strange values! Here is the output from sessionInfo().
>> sessionInfo()
> R version 2.6.0 (2007-10-03)
> i386-pc-mingw32
>
> locale:
> LC_COLLATE=English_United States.1252;LC_CTYPE=English_United
> States.1252;LC_MONETARY=English_United
> States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252
>
> attached base packages:
> [1] tools stats graphics grDevices utils datasets methods
> [8] base
>
> other attached packages:
> [1] beadarray_1.6.0 affy_1.16.0 preprocessCore_1.0.0
> [4] affyio_1.6.1 geneplotter_1.16.0 lattice_0.16-5
> [7] annotate_1.16.1 xtable_1.5-2 AnnotationDbi_1.0.6
> [10] RSQLite_0.6-4 DBI_0.2-4 Biobase_1.16.1
> [13] limma_2.12.0
>
> loaded via a namespace (and not attached):
> [1] grid_2.6.0 KernSmooth_2.22-21 RColorBrewer_1.0-2
>
>
>
>
>
>
>> 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,imageManipu
>>>>> la
>>>>> ti
>>>>> on="sharpen",
>>>>> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod="none",offse
>>>>> t=
>>>>> 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
>>>>>
>
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