[BioC] Illumina beadlevel expression data
Ina Hoeschele
inah at vt.edu
Fri Nov 16 15:16:45 CET 2007
Matt Ritchie wrote:
> 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).
>
yes, although that way I am getting quite a few (actually a lot) of
negative G values, then my only choices are to apply the 'minimum' or
'half' methods myself (but not others such as RMA that I wanted to try).
> 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.
>
the folder containing all files for the 12 arrays of one Beadchip are at
http://staff.vbi.vt.edu/inah/1814647013/
Thanks, Matt!
Ina
> 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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