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Illumina 450K analysis with a probe selection

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

methylation 450k illumina
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updated by andrew.j.skelton73 andrew.j.skelton73 • written by IOM • IOM

1 answer

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.

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written by andrew.j.skelton73 andrew.j.skelton73

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

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written by IOM • IOM

If an answer is helpful it's appropriate to upvote it. If the answer resolved your question, you should mark it as accepted.

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written by WouterDeCoster WouterDeCoster

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