Hi all!
Our lab doesn't have a lot of bioinformatics experience so over the last few months I have painfully reconstructed pipelines for cDNA and direct RNA Nanopore analysis. However, now we want to move into the RNA methylation space.
I've basecalled my output with dorado and aligned with dorado to generate a sorted bam file that contains MM tags and methylation frequencies. I can visualize this file in IGV and qualitatively identify our methylated residues, but I'd like to quantify this and have hit a wall.
Specifically, we'd like to look at differential methylation between the two major transcript isoforms of our gene of interest. We want to see if there are differences in methylation sites, and frequencies, between these isoforms that might affect their splicing. Additionally, we want to compare our gene of interest's methylation patterns to those of its 99.9% homologous partner gene.
I am relatively comfortable in R and thought I could accomplish this task there, but I'm not sure how the MM tags or methylation fractions are handled. I'd be very grateful if people could recommend tools or packages to use that will allow us to better visualize the methylation sites in our transcripts, and compare those sites and frequencies between gene isoforms or similar genes.
Thank you!
Nanopore offers
modkit: https://github.com/nanoporetech/modkitmeOW: https://github.com/mgaleyuw/MeOW
If you are familiar with epi2ME then it should also be useful: https://labs.epi2me.io/notebooks/Modified_Base_Tutorial.html.