Dogme: A nextflow pipeline for reprocessing nanopore RNA and DNA modifications

The Oxford Nanopore Technologies (ONT) platform allows for the direct detection of RNA and DNA modifications from unamplified nucleic acids, which is a significant advantage over other platforms. However, the rapid updates to ONT basecalling models and the evolving landscape of computational tools for modification detection bring about challenges for reproducible and standardized analyses. To address these challenges, we developed Dogme, which is a Nextflowbased workflow that automates the processing of ONT data, including basecalling, alignment, modification detection, and transcript quantification. Dogme automates the reprocessing of ONT POD5 files by integrating basecalling using Dorado, read mapping using minimap2 and subsequent analysis steps such as running modkit. The pipeline supports three major types of ONT sequencing data – direct RNA (dRNA), complementary DNA (cDNA), and genomic DNA (gDNA) – enabling comprehensive analyses across different library preparations. Dogme facilitates detection of diverse RNA modifications supported by Dorado such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, pseudouridine, 2’-Omethylation (Nm) and DNA methylation, while concurrently quantifying full-length transcript isoforms LR-Kallisto for transcript quantification for dRNA and cDNA.

Results

We applied Dogme to three separate mouse C2C12 myoblast replicates using direct RNA sequencing on MinION flow cells. We detected an average of 147,879 m6A, 86,673 m5C, 21,242 inosine, 24,540 pseudouridine, and 83,841 2’- O-methylation sites per replicate with 96,581 m6A, 43,446 m5C, 8,825 inosine, 10,048 pseudouridine, and 30,157 2’-O- methylation sites detected in all three biological replicates. The pipeline produced reproducible modification profiles and transcript expression levels across replicates, demonstrating its utility for integrative long-read transcriptomic and epigenomic analyses.

Availability

Dogme is implemented in Nextflow and is freely available under the MIT license at https://github.com/mortazavilab/dogme , with documentation provided for installation and usage.