Hi,
I am relatively new to the analysis of methylation data (Illumina 450k), and I have a question on how the methylation values are calculated in minfi. It did not became clear to me even after reading the help pages.
I am asking because for some probes I observe that both Meth and Unmeth values of the non-BG corrected and non-normalized data are exactly zero, whereas the Green and Red channels are not. As a result NaN's are returned for the beta values, which interferes with the clustering of the raw data (QC-ing).
- Could this be due to background correction, even though this should not be performed (i.e. this behavior is unintentional)??
- How to avoid these zero's? Likely by using an offset, but how to best do this?

Thanks for any clarification!
Guido


Example:
> rgSet <- read.450k.exp(base = myDir, targets = targets)
> rgSet
RGChannelSet (storageMode: lockedEnvironment)
assayData: 622399 features, 20 samples
  element names: Green, Red
phenoData
  sampleNames: PBMC_4 PBMC_13 ... PBMC_24 (20 total)
  varLabels: Sample_Name Sample_Group ... filenames (8 total)
  varMetadata: labelDescription
Annotation
  array: IlluminaHumanMethylation450k
  annotation: ilmn.v1.2
>
# Lets check the probeset on row 371,399, since that is one of 80 returning NaN's
# First extract Green and Red channel intensities for 1st 2 arrays:
> getGreen(rgSet)[371399,1:2]
PBMC_4 PBMC_13
    295     325
> getRed(rgSet)[371399,1:2]
PBMC_4 PBMC_13
    115     253
>
# Now convert into methylation signal without any BG correction and normalization
> mset <- preprocessRaw(rgSet)
> mset
MethylSet (storageMode: lockedEnvironment)
assayData: 485512 features, 20 samples
  element names: Meth, Unmeth
phenoData
  sampleNames: PBMC_4 PBMC_13 ... PBMC_24 (20 total)
  varLabels: Sample_Name Sample_Group ... filenames (8 total)
  varMetadata: labelDescription
Annotation
  array: IlluminaHumanMethylation450k
  annotation: ilmn.v1.2
Preprocessing
  Method: Raw (no normalization or bg correction)
  minfi version: 1.6.0
  Manifest version: 0.4.0
>
# Extract methylation values for indicated probeset for 1st two arrays:
>  getMeth(mset)[371399,1:2]
PBMC_4 PBMC_13
      0    2660
>  getUnmeth(mset)[371399,1:2]
PBMC_4 PBMC_13
      0     181
>
#^ Values=0 is returned, why is this?

# convert / extract betas:
> betas = getBeta(mset)
> betas[371399,1:2]
PBMC_4 PBMC_13
    NaN 0.93629
>
# NaN's are introduced...


> sessionInfo()
R version 3.0.0 Patched (2013-04-15 r62590)
Platform: x86_64-w64-mingw32/x64 (64-bit)

locale:
[1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252    LC_MONETARY=English_United States.1252 LC_NUMERIC=C
[5] LC_TIME=English_United States.1252

attached base packages:
[1] parallel  stats     graphics  grDevices utils     datasets  methods   base

other attached packages:
[1] IlluminaHumanMethylation450kmanifest_0.4.0 limma_3.16.3                               minfi_1.6.0
 [4] Biostrings_2.28.0                          GenomicRanges_1.12.2                       IRanges_1.18.1
 [7] reshape_0.8.4                              plyr_1.8                                   lattice_0.20-15
[10] Biobase_2.20.0                             BiocGenerics_0.6.0

loaded via a namespace (and not attached):
[1] beanplot_1.1          grid_3.0.0            illuminaio_0.2.0      MASS_7.3-26           matrixStats_0.8.1     mclust_4.1            multtest_2.16.0
 [8] nor1mix_1.1-4         preprocessCore_1.22.0 R.methodsS3_1.4.2     RColorBrewer_1.0-5    siggenes_1.34.0       splines_3.0.0         stats4_3.0.0
[15] survival_2.37-4       tools_3.0.0
>


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