Differential RNA modification calling at the isoform resolution using Nanopore direct RNA sequencing

Introduction

Mako is a bioinformatics pipeline designed for differential RNA modification calling at the isoform resolution using Nanopore direct RNA sequencing. It takes a samplesheet and POD5 files as input, performs basecalling and alignment, and then applies various statistical methods to identify differentially modified sites between experimental conditions. If you already have pre-basecalled data or m6Anet results, you can skip the basecalling step and directly analyze the modification data as well.

Mako will also produce visualisations for quality control and assessment.

Steps

1. Basecalling with dorado
2. Choice of modification caller:
   1. Dorado
   2. m6Anet
3. Site-level aggregation, filtering, and selection
4. Choice of differential analysis methods:
   1. Linear mixed-effects models
   2. Modkit
5. Visualization of results

Documentation

Please refer to the documentation for detailed instructions on how to use the pipeline, including input requirements, parameter settings, and output interpretation.

Installation

Read the quick start. But if you're in a rush:

# Clone the repository
git clone https://github.com/shimlab/mako && cd mako

# Test the pipeline
nextflow run main.nf --help
nextflow run main.nf -profile testing

Citations

Mako does not have a preprint yet. Please check back later for updates.

Contributors

This package is developed and maintained by Yulin Wu, Oliver Cheng, Sophie Wharrie and Heejung Shim in the Shim Lab at the University of Melbourne.

Please leave an issue if you find a bug or have a feature request. Thank you!