Agent skill
bio-long-read-sequencing-nanopore-methylation
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.
Install this agent skill to your Project
npx add-skill https://github.com/majiayu000/claude-skill-registry/tree/main/skills/data/nanopore-methylation
SKILL.md
Nanopore Methylation Calling
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 [email protected] \
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
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