Methylation Arrays

Martin Morgan (mtmorgan@fhcrc.org), Fred Hutchinson Cancer Research, Center, Seattle, WA, USA.
24 August 2014

E.g., HumanMethylation450k

  • Interrogate fragmented whole-genome DNA for methylationat specific genomic locations

Workflow

  1. Experimental design: replicate groups
  2. Wet-lab protocol
  3. beta: per-sample probe differential methylation
  4. Quality assessment
  5. Between-treatment differential methylation
  6. Interpretation

Illumina bead arrays

Methylation workflow http://en.wikipedia.org/wiki/Illumina_Methylation_Assay

Essential data

Probe x Sample matrix of beta differential methylation values

            R02C02 R04C01  R05C02 R04C02
cg00050873 0.91891 0.5759 0.95297 0.5423
cg00212031 0.09371 0.6548 0.14035 0.4636
cg00213748 0.80838 0.4773 0.70559 0.3176
cg00214611 0.08443 0.7704 0.17167 0.5501
cg00455876 0.78155 0.3345 0.75446 0.4835
cg01707559 0.09194 0.3903 0.07912 0.3732

'Metadata'

  • Associated with features (fData()) and samples (pData())
  • Important to coordinate with methylation values, e.g., subsetting
  • Solution: class to represent combined methylation and metadata

ExpressionSet

Example: 

library(minfi)
library(minfiData)
MsetEx

MethylSet (storageMode: lockedEnvironment)
assayData: 485512 features, 6 samples 
  element names: Meth, Unmeth 
phenoData
  sampleNames: 5723646052_R02C02
    5723646052_R04C01 ...
    5723646053_R06C02 (6 total)
  varLabels: Sample_Name Sample_Well
    ... filenames (13 total)
  varMetadata: labelDescription
Annotation
  array: IlluminaHumanMethylation450k
  annotation: ilmn12.hg19
Preprocessing
  Method: Raw (no normalization or bg correction)
  minfi version: 1.7.7
  Manifest version: 0.4.0

Example: pData

head(pData(MsetEx), 3)

                  Sample_Name Sample_Well
5723646052_R02C02    GroupA_3          H5
5723646052_R04C01    GroupA_2          D5
5723646052_R05C02    GroupB_3          C6
                  Sample_Plate Sample_Group
5723646052_R02C02           NA       GroupA
5723646052_R04C01           NA       GroupA
5723646052_R05C02           NA       GroupB
                  Pool_ID person age sex
5723646052_R02C02      NA    id3  83   M
5723646052_R04C01      NA    id2  58   F
5723646052_R05C02      NA    id3  83   M
                  status  Array     Slide
5723646052_R02C02 normal R02C02 5.724e+09
5723646052_R04C01 normal R04C01 5.724e+09
5723646052_R05C02 cancer R05C02 5.724e+09
                                                 Basename
5723646052_R02C02 ../extdata/5723646052/5723646052_R02C02
5723646052_R04C01 ../extdata/5723646052/5723646052_R04C01
5723646052_R05C02 ../extdata/5723646052/5723646052_R05C02
                                                filenames
5723646052_R02C02 ../extdata/5723646052/5723646052_R02C02
5723646052_R04C01 ../extdata/5723646052/5723646052_R04C01
5723646052_R05C02 ../extdata/5723646052/5723646052_R05C02

Example: subset

MsetEx[, pData(MsetEx)$status == "normal"]

MethylSet (storageMode: lockedEnvironment)
assayData: 485512 features, 3 samples 
  element names: Meth, Unmeth 
phenoData
  sampleNames: 5723646052_R02C02
    5723646052_R04C01
    5723646053_R05C02
  varLabels: Sample_Name Sample_Well
    ... filenames (13 total)
  varMetadata: labelDescription
Annotation
  array: IlluminaHumanMethylation450k
  annotation: ilmn12.hg19
Preprocessing
  Method: Raw (no normalization or bg correction)
  minfi version: 1.7.7
  Manifest version: 0.4.0

'S4' Classes

  • Similar to classes we have already seen
  • Different syntax for discovering methods, finding help
class(MsetEx)

showMethods("pData", where=search())
showMethods(class=class(MSetEx),
    where=search())

class ? MethylSet
method ? "getBeta,MethylSet"

Resources

  • minfi landing page and vignette browseVignettes("minfi")
  • Biobase vignette “An Introduction to Biobase and ExpressionSet”, browseVignettes("Biobase")

Acknowledgements

Funding

  • US NIH / NHGRI 2U41HG004059; NSF 1247813

People

  • Seattle Bioconductor team: Sonali Arora, Marc Carlson, Nate Hayden, Valerie Obenchain, Hervé Pagès, Dan Tenenbaum
  • Vincent Carey, Robert Gentleman, Rafael Irizzary, Sean Davis, Kasper Hansen, Michael Lawrence, Levi Waldron