Investigating differential modification on native RNA (direct RNA sequencing)

Modkit contains two functions for investigating methylation in direct RNA sequencing reads. These two subcommands, dmr isoform and dmr compare-tx-sites, are meant to use a bedMethyl generated by pileup starting from a modBAM containing direct RNA reads aligned to a reference transcriptome, as opposed to a reference genome.

There are detailed instructions for how to run pileup in that command’s page. Succinctly, a command similar to the one below will generate a bedMethyl table suitable for downstream use.

$ modkit pileup $modbam $output_pileup.bed.gz --modified-bases m6A --ref $transcriptome.fa --preload-references --bgzf
$ tabix $output_pileup.bed.gz

The following tools are available:

1. Compare modifications between isoforms of genes
2. Compare modifications between two conditions, aggregating isoform modifications on the gene level