[BioC] MEDIPS: how does MEDIPS define a methylated region / cluster?

Sun Sep 7 20:10:58 CEST 2014

Dear Lukas,
Again, I am so grateful for your quick reply.

The only manual for MEDIPS that I know of is this one: http://www.bioconductor.org/packages/release/bioc/manuals/MEDIPS/man/MEDIPS.pdf
and I have looked and just can't find the section 7.7 that you mentioned.

The reason I asked about how MEDIPS defines a cluster is because I wanted to try and reduce the number of methylated regions that I look at and then use these "consolidated" regions to do a principal component analysis (PCA). I have 12 cell types and I wanted to see if I could either do a PCA or Unsupervised Hierarchical Clustering to separate these 12 cell types into subgroups, based on their methylation profile.

You said: "When windows with significant differential enrichment between groups have been identified, the MEDIPS.mergeFrames() function can be used to merge adjacent significant windows into extended regions."
But at this point, I don't have any groupings. Normally, one would have a control and test group and so you can do what you suggested. But I have only, for example, a test group and I want to know if this test group can be further subdivided into a few subgroups based on their methylation profile. I would think that it would be computationally intensive if I used original 100bp windows generated by MEDIPS and entered all the rms values for each window of each cell type into the PCA.

You said: "Instead of merging any values across windows, I recommend to make use of the MEDIPS.createROIse() function which allows for analyzing specific regions of interest."
I didn't want my PCA analysis to be biased by a predefined "region of interest", be it whether the ROI is a promoter, CpG, etc. I wanted to just input the raw methylation scores (rms) to see what I could get. So that is why I asked if the rms can be added up (in question 4 of my last email)...also I was curious if it could be done.

Once again., thanks so very much for taking time from your busy schedule to answer my questions.

Best regards,
Allen

________________________________________
From: Lukas Chavez [lukas.chavez.mailings at googlemail.com]
Sent: Saturday, September 06, 2014 11:09 AM
To: Allen [guest]
Cc: bioconductor at r-project.org; Chong Kim San Allen; MEDIPS Maintainer
Subject: Re: [BioC] MEDIPS: how does MEDIPS define a methylated region / cluster?

Dear Allen,

please see my comments below. Moreover, please consider reading section 7.7 of the MEDIPS manual.

Best,
Lukas

On Fri, Sep 5, 2014 at 1:14 AM, Allen [guest] <guest at bioconductor.org<mailto:guest at bioconductor.org>> wrote:
Hi,
I have aligned my sequencing data for one sample (filename: Ca2_MAPQ20.bam --- because I filtered out everything with MAPQ20 or less) and I've put it through MEDIPS. At the moment, I am not making a comparison between samples. Therefore, the result file shows the methylation profile for only one sample. In this results file, I noticed that the data is organized as such:
chr     start   stop    CF      Ca2_MAPQ20.bam.counts   Ca2_MAPQ20.bam.rpkm     Ca2_MAPQ20.bam.rms      Ca2_MAPQ20.bam.prob     MSets1.counts.mean      MSets1.rpkm.mean        MSets1.rms.mean MSets1.prob.mean

1) I tried reading the Down et al. (2008) paper that explains the concept of coupling factor and I think it is, simply put, a measure of local CpG density. I am not sure if my understanding of 'coupling factor' is correct?

That is correct.

2) This lead to question how or what MEDIPS defines as a "region or cluster"? That is to say, on Chromosome 1, I have reads aligning from position "1002501" to "1003300" but then there is a region of 1400 bp (from "1003301" to "1004700") where there are no reads aligned (rpkm = 0). Then, again, from "1004701" to "1005400", there reads aligning to this region. So my question is does MEDIPS consider this to be a case of 2 methylated regions, that is, "1002501-1003300" and "1004701-1005400" or does MEDIPS try to consolidate these 2 methylated regions into one methylated region/cluster (that is, from "1002501-1005400") since they are fairly close to one another.

Each window is considered as one region; CpG density, counts, rpkm, rms, and differential coverage will be calculated for each window separately. When windows with significant differential enrichment between groups have been identified, the MEDIPS.mergeFrames() function can be used to merge adjacent significant windows into extended regions.

3) I used "uniq=TRUE" to get rid of stacked/clonal reads and so for each 100 bp bin, I am usually getting rpkm=1 within each bin but occasionally, it may be as many rpkm=3. This lead me to wonder how the relative methylation score ("Ca2_MAPQ20.bam.rms") is calculated? For example, 3 bins have a value of 1 rpkm each, but one has an rms value of "1660.409928", another an rms of "7122.10254" and the third is only "679.1814523". How is that possible that 3 bins that have the same rpkm can have such varying rms values?

This is due to the different CpG densities in these windows.

4)Can the rms be added up for a region so as to represent the cumulative methylation level for that region. I am asking because I do not know if the rms value is a log value or what? So, in my above question (3), if the 3 bins are adjacent to one another and I decide to cluster them together and consider them as one methylated region, then would the rms value for this new 300bp bin that I have created be 1660.409928 + 7122.10254 + 679.1814523 = 9461.68.

Instead of merging any values across windows, I recommend to make use of the MEDIPS.createROIse() function which allows for analyzing specific regions of interest.

5)Finally, what does "Ca2_MAPQ20.bam.prob" represent? I figured that is some sort of a probability score but I am not sure of what.

As mentioned in the manual, please disregard the prob values which will be removed in a future version.

Thanks in advance for any assistance in answering my questions.

Best regards,
Allen

 -- output of sessionInfo():

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