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

TL;DR

Dogme is a pipeline that automates the analysis of RNA and DNA modifications using Nanopore sequencing data, enabling reproducible and standardized results.

Contribution

Dogme introduces an automated pipeline for reprocessing Nanopore RNA and DNA modification data using Nextflow, integrating basecalling, alignment, and modification detection.

Findings

Dogme detected thousands of RNA modification sites across three mouse C2C12 myoblast replicates.

The pipeline produced reproducible modification profiles and transcript expression levels across replicates.

Dogme supports diverse modification types including m6A, m5C, inosine, pseudouridine, and Nm.

Abstract

Oxford Nanopore (ONT) sequencing 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 to automate 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 sequencing data—direct RNA (dRNA), complementary DNA (cDNA), and genomic DNA (gDNA). Dogme facilitates detection of diverse RNA modifications supported by Dorado such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), inosine, pseudouridine, 2’-O-methylation (Nm) and DNA methylation, while concurrently quantifying full-length transcript isoforms LR-Kallisto for transcript quantification for dRNA and cDNA.

We applied Dogme to three separate mouse C2C12 myoblast replicates using direct RNA sequencing on MinION flow cells. We detected 96 603 m6A, 43 476 m5C, 8829 inosine, 10 055 pseudouridine, and 30 320 Nm sites in 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.

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.

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