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

Area of Science:

• Genomics
• Epigenomics
• Bioinformatics

Background:

• Oxford Nanopore Technologies (ONT) enables direct RNA and DNA modification detection from unamplified nucleic acids.
• Rapid updates in ONT basecalling models and computational tools challenge reproducible analysis.
• Standardized and automated workflows are needed for accurate modification detection and transcript quantification.

Purpose of the Study:

• To develop Dogme, a Nextflow-based workflow for automated processing of ONT data.
• To enable reproducible detection of RNA and DNA modifications and transcript quantification.
• To support direct RNA (dRNA), complementary DNA (cDNA), and genomic DNA (gDNA) sequencing data.

Main Methods:

• Dogme automates ONT POD5 file processing, including basecalling with Dorado and read mapping with minimap2.
• Integrates modification detection using modkit for various RNA modifications (m6A, m5C, inosine, pseudouridine, Nm) and DNA methylation.
• Performs full-length transcript isoform quantification using LR-Kallisto for dRNA and cDNA data.

Main Results:

• Applied Dogme to mouse C2C12 myoblasts using direct RNA sequencing.
• Detected thousands of RNA modification sites (m6A, m5C, inosine, pseudouridine, 2'-O-methylation) across replicates.
• Demonstrated reproducible modification profiles and transcript expression levels, highlighting utility for integrative analyses.

Conclusions:

• Dogme provides a robust and reproducible solution for analyzing ONT sequencing data.
• Facilitates comprehensive transcriptomic and epigenomic studies using long-read sequencing.
• The workflow is freely available, promoting standardization in the field.