[BioC] Illumina beadlevel expression data
Matt Ritchie
Matt.Ritchie at cancer.org.uk
Fri Nov 16 15:53:19 CET 2007
Dear Ina,
Thanks for the data. There appears to be a quality problem with these
arrays that occurred during scanning. The registration scores in the
Metrics.txt file indicates that the images have not registered properly (the
RegGrn values should be around 1 for a good scan - in your case they are
around 0.14). As a result, many beads have zero intensities. Please check
with the people who generated this data to determine what went wrong.
Best wishes,
Matt
On 16/11/07 14:16, "Ina Hoeschele" <inah at vt.edu> wrote:
> 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,imageMani
>>>>>>> pu
>>>>>>> la
>>>>>>> ti
>>>>>>> on="sharpen",
>>>>>>> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod="none",off
>>>>>>> se
>>>>>>> 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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