How to extract CpG level for IGV plot

[abearab]

Hi @FelixKrueger – I have another question! I could solve my issue here #633 and now I'm trying to show a close-ups of genomic region of interest, something like this plot:

Fig 2F. https://doi.org/10.1016/j.cell.2021.03.025 (@hovestadt's original work)

(F) A comparison of CpG methylation along a 55-kb window that includes the CLTA locus. Tracks labeled “Untr.” represent untransfected cells; the “NT” tracks represent cells transfected with CRISPRoff and non-targeting sgRNA; and the “T” tracks represent cells transfected with CRISPRoff and targeting sgRNA. R1 and R2 represent two technical replicates. Red marks represent methylated (beta-value >0.5) and the blue marks represent unmethylated (<0.5) CpG dinucleotides. CpG islands are shown in green.

Close-ups of genomic regions were generated by visualizing beta-values of individual loci in IGV. Data was displayed as bar charts (min/0: blue, mid/0.5, max/1: red).

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I don't know the right way to prepare the CpG methylation info from bismark outputs for building this visualization. Do you have any recommendation? Thanks agin for sharing your expert advice.

[FelixKrueger]

I am not exactly sure about how to get the data into IGV as we have used Seqmonk for this purpose in the past, see e.g. here: https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=4965689_f1000research-5-9980-g0002.jpg

Here are some more examples on how to get started with methylation analysis in SeqMonk: https://www.bioinformatics.babraham.ac.uk/training.html#bsseq. It is really dead-easy as SeqMonk simply accepts Bismark coverage files (.cov.gz) as input, so you can literally get a figure as the one shown above in the matter of a few minutes.

[abearab]

Thanks for your response! One dumb question, the expected .cov.gz file is the output of bismark_methylation_extractor command, right? How would you use it (i.e. what options)?

[FelixKrueger]

It is indeed, just use the option --bedGraph for the methylation extraction. Or --help for more info. Here are the docs: https://felixkrueger.github.io/Bismark/bismark/methylation_extraction/

[abearab]

Okay, that's the bedGraph file. Thanks

[FelixKrueger]

The coverage file is pretty much the same as the bedGraph file, but in addition contains the counts methylated and unmethylated cytosines.