ONT Methylation calling

Question

[Franci]

I HAVE RECENTLY USED OXFORD NANOPORE SEQUENCING FOR A 960bp DNA containing methylated CpGs. While setting up the sequencing i have selected modified base-5mC and also loaded a reference fasta file. Post sequencing i have got .fastq, .pod5, .bam files. Do the .bam files contain methylation calling info? If so, how can i visuallize using igv? currently when i upload on igv, and select Color alignment by > Bisulfite mode> CG , all my Cs and Gs are highlighted red. Is it correct visualization? Do i need to do methylation calling by megalodon separately or MinKNOW software does it when i choose modified base?

Answer 1

[colindaven]

You can have a look at this tutorial for the basics. https://epi2me.nanoporetech.com/mod-validation-data/

Don't use megalodon, use modkit. You need to make sure the BAM file contains the MM and ML tags too, else no modification info is present there.

[Franci]

Thank you. I will definitely try this. Could you also tell me if i can directly upload the .bam files generated by MinKNOW for IGV visualization?

[colindaven]

I never tried this so don't know, but you can try it.