Illumina 450K analysis with a probe selection

Question

[IOM]

Hi,

Thanks for reading, I would like to ask you for your opinion. I would like perform a methylation analysis comparing two groups of samples with a determined number of probes/genes, not using all the probes available in the array (this gene list comes from a literature search). My questions are:

• Should probe selection be done before the contrast? Reduce the number of probes to a few hundreds/thousands before any comparison.
• Should probe selection be done after the contrast? Use all the probes and do a heatmap, for example, only with selected probes.
• What statistical consequences any of this selections would have?
• Does anybody know how to perform the probe selection in a workflow using minfi/limma, for example? I am asking this because the annotation of the probes is done after performing the contrasts.

Any help will be much appreciated. Thanks

IOM

Answer 1

[andrew.j.skelton73]

Assuming that you've applied a normalisation process with Minfi, and you have a resulting GenomicRatioSet class object, you can get the annotation using:

norm_data_f         <- norm_data_f %>% addSnpInfo
norm_data_f         <- norm_data_f %>% dropLociWithSnps(., snps = c("SBE","CpG","Probe"), maf  = 0.05)
annotation          <- norm_data_f %>% getAnnotation %>% as.data.frame

Which gives you a full set of annotation for your probes, prior to modelling with Limma. The GenomicRatioSet is also a subsettable S4 class, so you can use the annotation to provide sensible filtering. Minfi provides a nice helper function dropLociWithSnps for probe level filtering.

Generally speaking, I wouldn't cherry pick genes to test, I'd run the contrast across all probes passing your probe level filters. Pre-selecting genes to test means that you reduce the total number of tests, and the resulting FDR is more lenient.

[IOM]

Hi Andrew,

Thanks very much for your response. As you say, reducing the number is not appropriate and I would expect those genes to be differentially methylated using all probes if they truly were (without the reduction), is that correct? Thanks

IOM

[andrew.j.skelton73]

That is correct, yes.

[IOM]

Cheers

[WouterDeCoster]

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