/bio-long-read-sequencing-nanopore-methylation

name: bio-long-read-sequencing-nanopore-methylation
description: Calls DNA methylation from Oxford Nanopore sequencing data using signal-level analysis. Use when detecting 5mC or 6mA modifications directly from nanopore reads without bisulfite conversion.
tool_type: cli
primary_tool: modkit

Version Compatibility

Reference examples tested with: methylKit 1.28+, minimap2 2.26+, samtools 1.19+

Before using code patterns, verify installed versions match. If versions differ:

• CLI: `<tool> --version` then `<tool> --help` to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Nanopore Methylation Calling

**"Call methylation from my Nanopore reads"** → Extract 5mC/6mA modification probabilities from basecalled reads and summarize per-site methylation frequencies.

• CLI: `modkit pileup aligned.bam methylation.bed --ref ref.fa`

Modern Workflow (modkit)

ONT's modkit is the recommended tool for methylation analysis from basecalled data.

Extract Methylation from BAM

# Assumes BAM has MM/ML tags from dorado basecalling
modkit pileup input.bam methylation.bed \
    --ref reference.fa \
    --cpg \
    --combine-strands

Output Format

# bedMethyl format
chr1  1000  1001  .  10  +  1000  1001  0,0,0  10  80.5
# Columns: chrom, start, end, name, score, strand, thickStart, thickEnd,
#          itemRgb, coverage, percent_modified

Basecalling with Methylation

# Dorado basecalling with 5mC model
dorado basecaller dna_r10.4.1_e8.2_400bps_sup@v4.2.0 \
    pod5_dir/ \
    --modified-bases 5mCG \
    > calls.bam

# Index and align
samtools fastq calls.bam | \
    minimap2 -ax map-ont -y reference.fa - | \
    samtools sort -o aligned.bam
samtools index aligned.bam

Region-Specific Analysis

# CpG islands only
modkit pileup aligned.bam cpg_islands.bed \
    --ref reference.fa \
    --cpg \
    --include-bed cpg_islands.bed

# Promoter regions
modkit pileup aligned.bam promoters.bed \
    --ref reference.fa \
    --cpg \
    --include-bed promoters.bed

Sample Summary

# Get modification summary statistics
modkit summary aligned.bam

# Output includes:
# - Total reads with modifications
# - Modification types detected
# - Fraction modified per type

Differential Methylation

# Create BED files for each sample
modkit pileup sample1.bam sample1.bed --ref ref.fa --cpg
modkit pileup sample2.bam sample2.bed --ref ref.fa --cpg

# Compare with methylKit or DSS in R

Quality Considerations

• Minimum coverage: 10x for reliable calls
• Modified base probability threshold: 0.5 default, adjust as needed
• Combine strands for CpG (symmetric methylation)

Related Skills

• long-read-sequencing/basecalling - Dorado basecalling
• methylation-analysis/methylation-calling - General methylation concepts
• methylation-analysis/dmr-detection - Differential methylation