how extract CpG methylation calls from nanopore long sequencing bam file ?

Question

[reziyamaimaiti0331]

I already have 6mA methylized sorted bam file and i want to make plot enrichment profile with 6mA and mCpG methylation fraction ,so how extract CpG methylatiuon calls from nanopore long sequencing bam file ?

Answer 1

[GenoMax]

Do you know if your file already has the methylation calls for mCpG? If they are there then you can use modkit (LINK) provided by ONT.

Otherwise you will have to go back and re-basecall methylated bases with dorado and pod5 files from original data folder, followed by the analysis above.

[reziyamaimaiti0331]

thank you so much ,your information really so helpfull and already solved problem ,another things i want to ask is i am running basecalling using epi2me and nextflow in linux ,but there is always error and i did not understand it .:ERROR ~ Error executing process > 'wf_dorado:dorado (2)'

Caused by: Failed to pull singularity image command: singularity pull --name ontresearch-dorado-shae9327ad17e023b76e4d27cf287b6b9d3a271092b.img.pulling.1754057268435 docker://ontresearch/dorado:shae9327ad17e023b76e4d2 7cf287b6b9d3a271092b > /dev/null status : 255 hint : Try and increase singularity.pullTimeout in the config (current is "20m") message:

Answer 2

[Istvan Albert]

The BAM file has tags that contain the information you need.

There are tags such as:

• MM: Modified base motif and positions.
• ML: Per-base modification likelihoods

You can use tools like

• https://github.com/epi2me-labs/modbam2bed

or write a program with PySAM to extract that information

[colindaven]

modkit is the updated version of modbam2bed, which is deprecated. Megalodon is also deprecated.

So use dorado -> minimap -y -> modkit.