GPTomics/bio-long-read-sequencing-nanopore-methylation
long-read-sequencing/nanopore-methylation/SKILL.md
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 --global
skillsauthnpx skillsauth add GPTomics/bioSkills bio-long-read-sequencing-nanopore-methylationInstall this skill globally with one command. Works with Claude Code, Cursor, and Windsurf.
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SKILL.md
- 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> --versionthen<tool> --helpto 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 [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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