usage-guide.md

Nanopore Methylation Calling Usage Guide

Overview

This guide covers detecting DNA methylation directly from Oxford Nanopore sequencing data.

Prerequisites

# modkit (ONT tool)
# Download from https://github.com/nanoporetech/modkit

# Dorado basecaller with methylation models
# Download from https://github.com/nanoporetech/dorado

# Supporting tools
conda install -c bioconda minimap2 samtools

Quick Start

Tell your AI agent what you want to do:

• "Call 5mC methylation from my nanopore BAM file"
• "Generate a methylation bedMethyl file from my ONT data"
• "Compare methylation between tumor and normal samples"
• "Extract methylation in promoter regions"

Example Prompts

Basic Methylation Calling

"Run modkit pileup on my aligned BAM to get CpG methylation calls"

"Generate a bedMethyl file combining both strands for CpG sites"

Region Analysis

"Extract methylation levels in CpG islands from my nanopore data"

"Calculate average methylation in gene promoters"

Differential Analysis

"Compare methylation between my two samples and find differentially methylated regions"

What the Agent Will Do

1. Verify BAM contains methylation tags (MM/ML)
2. Run modkit pileup with appropriate settings
3. Filter by coverage and quality thresholds
4. Generate bedMethyl output
5. Summarize methylation statistics

Tips

• Ensure basecalling used methylation-aware model
• Minimum 10x coverage recommended for reliable calls
• Combine strands for CpG (--combine-strands)
• Use --cpg flag to limit to CpG context
• Check modkit summary before analysis
• For 6mA detection, use appropriate model and flags