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

Area of Science:

• Genomics and Epigenomics
• Computational Biology
• Molecular Biology

Background:

• Oxford Nanopore (ONT) sequencing enables direct detection of RNA and DNA modifications.
• Rapid updates in ONT basecalling and analysis tools pose challenges for reproducible research.
• A need exists for standardized computational pipelines to analyze ONT data.

Purpose of the Study:

• To develop Dogme, an automated pipeline for basecalling, alignment, modification detection, and transcript quantification from ONT sequencing data.
• To support direct RNA (dRNA), complementary DNA (cDNA), and genomic DNA (gDNA) sequencing data.
• To facilitate the detection of diverse RNA modifications (e.g., m6A, m5C, inosine, pseudouridine, Nm) and DNA methylation.

Main Methods:

• Dogme is implemented in Nextflow, integrating tools like Dorado for basecalling, minimap2 for mapping, and modkit for modification analysis.
• The pipeline automates the reprocessing of ONT POD5 files.
• LR-Kallisto is used for transcript quantification of dRNA and cDNA data.

Main Results:

• Application of Dogme to mouse C2C12 myoblast direct RNA sequencing identified numerous modification sites (e.g., 96,603 m6A, 43,476 m5C).
• The pipeline demonstrated reproducible modification profiles and transcript expression levels across biological replicates.
• Results highlight Dogme's utility for integrated long-read transcriptomic and epigenomic analyses.

Conclusions:

• Dogme provides a robust and reproducible solution for analyzing ONT sequencing data.
• The pipeline streamlines complex analyses, enabling deeper insights into RNA and DNA modifications.
• Dogme is freely available, promoting standardization and accessibility in epigenomic research.