[BioC] Illumina BeadChips and beadarray

Matt Ritchie Matt.Ritchie at cancer.org.uk
Tue Aug 26 21:31:31 CEST 2008


Dear Ina,

Thanks for the further detail.  The intensities do look too low, especially
when you use the tifs to reconstruct the bead-level intensities.

If you put the tif and text files for these arrays online somewhere, I'll
get someone to look into this in more detail.  In the meantime, try
analysing the data you get from running readIllumina() with useImages=FALSE,
as it looks more reasonable than the data you are getting with
useImages=TRUE.

Best wishes,

Matt

> Hi Matt,
>    I've done all of the below and much more. I have also had our Illumina
> representative look at the data and he claims that everything is fine. Below
> are selected quantiles of the bead-level intensities (0.05, 0.25, 0.5, 0.75,
> 0.95) for one chip and its 12 strips.
> (1) using Illumina Grn values from txt files:
>> quantiles
>       [,1] [,2] [,3] [,4]    [,5]
>  [1,]   -1    0    0   11  418.00
>  [2,]   -1    0    0   12  447.00
>  [3,]   -2    0    6   80 1364.00
>  [4,]   -2    0    7   90 1409.45
>  [5,]    0    0    0    6  297.00
>  [6,]    0    0    0    5  286.00
>  [7,]   -1    0    4   69 1195.00
>  [8,]   -2    0    5   78 1301.00
>  [9,]    0    0    0    4  251.00
> [10,]    0    0    0    6  298.00
> [11,]   -1    0    4   62 1158.00
> [12,]   -2    0    5   73 1210.00
> 
> (2) using beadarray G values with backgroundMethod=subtract (same as none):
>> quantiles
>             [05]       [25] [50]         [75]      [95]
>  [1,]  -5.954797 -0.5736889    0  0.001486963  76.50805
>  [2,]  -6.716048 -0.6553347    0  0.000000000  74.87552
>  [3,]  -8.874994 -1.0853280    0 12.898865185 257.36723
>  [4,]  -9.878324 -1.1983333    0 12.881387407 248.49933
>  [5,]  -5.474805 -0.4840000    0  0.000000000  44.99409
>  [6,]  -5.847878 -0.4500096    0  0.000000000  36.67622
>  [7,] -10.023832 -1.1582759    0  7.222904311 198.11332
>  [8,]  -9.920325 -1.1513581    0  9.187925926 218.21951
>  [9,]  -5.433433 -0.3801279    0  0.000000000  32.71187
> [10,]  -5.631454 -0.4804800    0  0.000000000  43.97248
> [11,]  -9.375240 -1.0264076    0  6.900505556 200.94659
> [12,]  -9.692831 -1.1042407    0  8.113128889 205.05368
> 
> (3) using beadarray Grn values with backgroundMethod=normexp:
>> quantiles
>            [,1]      [,2]      [,3]      [,4]      [,5]
>  [1,]  6.157799  9.222023  9.625428  9.626493  84.89528
>  [2,]  7.833909 10.908199 11.311446 11.311446  83.60846
>  [3,]  9.575426 14.956604 15.860443 28.092912 272.54870
>  [4,]  8.655667 14.778997 15.819756 28.264072 263.87804
>  [5,] 11.174887 13.368365 13.607286 13.607286  52.19423
>  [6,]  5.395829  8.226291  8.523353  8.523353  43.72268
>  [7,]  8.578752 14.192817 15.117620 21.596692 212.34225
>  [8,]  8.297437 14.184411 15.152457 23.788444 232.78697
>  [9,]  6.201761  8.595434  8.813951  8.813951  38.92746
> [10,]  4.972100  7.794981  8.128802  8.128802  50.99212
> [11,]  8.195206 13.696098 14.540742 20.851138 214.80211
> [12,] 13.007751 17.648962 18.359116 24.356513 219.90004
> 
> Here are summaries for the summarized data:
> 
> (1) Illumina Grn bead values from txt files, summary-method = Illumina:
>> summary
>       Min.    1st Qu.     Median       Mean    3rd Qu.       Max.
>   -26.7600     0.6667     1.6800   104.9000     6.7400 32190.0000
>    -2.4750     0.7083     3.1140   297.7000    56.6200 29640.0000
>   -20.5900     0.4651     1.5860   155.8000     7.5650 31930.0000
>   -11.0800     0.6562     2.9700   298.3000    56.6000 28770.0000
>   -54.7200     0.4667     1.3850   112.1000     5.3750 31020.0000
>    -5.5780     0.6875     3.0310   320.9000    63.5900 28990.0000
>  
> (2) backgroundMethod=subtract, summary-method = Illumina:
>> summary
>      Min.   1st Qu.    Median      Mean   3rd Qu.      Max.
> -2.75800  -0.29720  -0.15130  -0.07024  -0.06051 167.00000
> -4.9880 -0.9490 -0.5366 -0.4486 -0.1624 46.3600
> -4.54900  -0.13050  -0.05114   0.95690  -0.01015 188.30000
> -5.2030 -0.9115 -0.5194 -0.5233 -0.2052 56.3800
> -13.060000  -0.111400  -0.038540   1.166000  -0.004997 172.300000
> -5.3580 -0.8505 -0.4745 -0.4903 -0.1856 39.3200
> 
> (3) backgroundMethod=normexp, summary-method = Illumina:
>> summary
>   8.652   9.946  10.160  10.160  10.370  12.450
>   12.19   14.93   15.30   15.37   15.62   61.94
>    7.72   10.49   10.97   11.03   11.49   15.02
>   11.66   14.27   14.62   14.61   14.90   40.31
>   7.201   8.174   8.291   8.282   8.392  26.040
>   12.61   15.60   16.16   16.24   16.80   44.74
> 
> 
> Where I really see a problem is with the MA plots of the unnormalized
> beadsummary data (see attached plots):
> (1) For the summaries created from the Illumina Grn values from txt files, MA
> plots look normal to me.
> (2) For the summaries created from the beadarray G values using
> backgroundMethod=normexp, the MA plots do NOT look as expected to me.
> 
> Can I upload some of the .txt and .tif files for you somewhere, if you are
> still willing to look at them?
> 
> MANY thanks, Ina
> 
> 
> ----- Original Message -----
> From: "Matt Ritchie" <Matt.Ritchie at cancer.org.uk>
> To: "Ina Hoeschele" <inah at vbi.vt.edu>
> Cc: bioconductor at stat.math.ethz.ch
> Sent: Monday, August 25, 2008 11:16:02 PM GMT -05:00 US/Canada Eastern
> Subject: Re: [BioC] Illumina BeadChips and beadarray
> 
> Dear Ina,
> 
> I can't see anything wrong with your commands (although you might find
> 
> cor(exprs(BSData.Illumina.txt.013), method="pearson")
> 
> is an easier way of getting the correlation between pairs of arrays).
> 
> Are there any quality issues with this data set that could be driving this
> unusual result?  Try plotting the raw data from BLData.Illumina.txt.013 and
> BLData.sharpen.normexp.txt.tif.013.  You could also try running
> 
> summary(getArrayData(BLData.Illumina.txt.013, what="G", array=1, log=FALSE))
> summary(exprs(BSData.Illumina.txt.013))
> summary(getArrayData(BLData.sharpen.normexp.txt.tif.013, what="G", array=1,
> log=FALSE))
> summary(exprs( BSData.sharpen.normexp.txt.tif.013))
> 
> to check that both the raw and summarised intensities are sensible (they
> should be on mostly positive and on the range 0 - 80000 (or so) if you have
> run getArrayData() and createBeadSummaryData() with log=FALSE).  If this
> doesn't turn up anything, perhaps you can send me a few .txt and .tif files
> from this experiment off list so that I can take a closer look at what is
> going on.
> 
> Best wishes,
> 
> Matt
> 
>> Hi Matt and others,
>>      I am working with Illumina beadlevel expression data.  When I read the
>> foreground intensities as provided by Illumina in the .txt files (Grn
>> column),
>> then perform beadsummary and then calculate Pearson correlation coefficients
>> between the six samples on one chip, I get correlations between .68 and .96.
>> However, when I read from the .tif files and use backgroundMethod = normexp
>> or
>> subtract, then perform beadsummary and again calculate Pearson correlation
>> coefficients, then I only get values that are essentially zero!
>> Here is the code that I use:
>> 
>> For reading from .txt files:
>> 
>> BLData.Illumina.txt.013 = readIllumina(textType=".txt",
>> arrayNames = c("1814647013_A_1","1814647013_A_2","1814647013_B_1",
>> "1814647013_B_2","1814647013_C_1","1814647013_C_2","1814647013_D_1",
>> "1814647013_D_2","1814647013_E_1","1814647013_E_2","1814647013_F_1",
>> "1814647013_F_2"), singleChannel=TRUE, useImages=FALSE,
>> normalizeMethod="none", backgroundMethod="none")
>> BSData.Illumina.txt.013 <-
>> 
createBeadSummaryData(BLData.Illumina.txt.013,log=FALSE,n=3,imagesPerArray=2,>>
w
>> hat="G",method="illumina")
>> Gvec1.13 <- NULL
>> Gvec1.13 <- exprs(BSData.Illumina.txt.013)[,1]
>> Gvec2.13 <- NULL
>> Gvec2.13 <- exprs(BSData.Illumina.txt.013)[,2]
>> Gvec3.13 <- NULL
>> Gvec3.13 <- exprs(BSData.Illumina.txt.013)[,3]
>> Gvec4.13 <- NULL
>> Gvec4.13 <- exprs(BSData.Illumina.txt.013)[,4]
>> Gvec5.13 <- NULL
>> Gvec5.13 <- exprs(BSData.Illumina.txt.013)[,5]
>> Gvec6.13 <- NULL
>> Gvec6.13 <- exprs(BSData.Illumina.txt.013)[,6]
>> Gvec13 <- cbind(Gvec1.13,Gvec2.13,Gvec3.13,Gvec4.13,Gvec5.13,Gvec6.13)
>> cor(Gvec13,method="pearson")
>> 
>> For reading from .tif files:
>> 
>> BLData.sharpen.normexp.txt.tif.013 = readIllumina(textType=".txt",
>> arrayNames = c("1814647013_A_1","1814647013_A_2","1814647013_B_1",
>> "1814647013_B_2","1814647013_C_1","1814647013_C_2","1814647013_D_1",
>> "1814647013_D_2","1814647013_E_1","1814647013_E_2","1814647013_F_1",
>> "1814647013_F_2"), singleChannel=TRUE, useImages=TRUE,
>> beadInfo=NULL, targets=NULL, storeXY=TRUE,
>> imageManipulation="sharpen", metrics=TRUE, metricsFile="Metrics.txt",
>> backgroundMethod="normexp", normalizeMethod="none")
>> BSData.sharpen.normexp.txt.tif.013 <-
>> 
createBeadSummaryData(BLData.sharpen.normexp.txt.tif.013,log=FALSE,n=3,images>>
P
>> erArray=2,what="G",method="illumina")
>> Š as above
>> 
>> What am I doing wrong with the reading/processing of the .tif files?
>> 
>> Thanks again, Ina



More information about the Bioconductor mailing list