Introduction
Mako is a bioinformatics pipeline designed for the differential analysis of RNA modifications between two groups using Oxford Nanopore Technologies (ONT) RNA004 direct RNA sequencing data. 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.
The software is written in Nextflow and utilises Docker/Singularity containerisation for reproducibility and ease of installation.
See Getting Started for instructions on how to install and run the pipeline.
Steps of the pipeline
- Basecalling with dorado
- Choice of modification caller:
- Dorado
- m6Anet
- Site-level aggregation, filtering, and selection
- Choice of differential analysis methods:
- Linear mixed-effects models
- Modkit
- Visualization of results