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Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Published on: June 3, 2019

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

Elnaz Abdollahzadeh¹, Ali Mortazavi²

¹Department of Developmental and Cell Biology, UC Irvine, Irvine, CA 92697, United States.

Bioinformatics (Oxford, England)

|March 2, 2026

Summary

Dogme is a new pipeline that automates Oxford Nanopore sequencing data analysis for RNA and DNA modifications. It ensures reproducible results for direct RNA, cDNA, and genomic DNA sequencing, enabling comprehensive transcriptomic and epigenomic insights.

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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.