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Run npx skillmds add majiayu000/bio-long-read-sequencing-nanopore-methylation in your terminal (requires Node.js), paste this page's agent-chat prompt into Claude, Cursor, or any MCP-connected agent, or download the SKILL.md file and copy it into your agent's skills directory.

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Is Bio Long Read Sequencing Nanopore Methylation free to use?

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Who published Bio Long Read Sequencing Nanopore Methylation?

majiayu000 (@majiayu000) published this skill. Their other Agent Skills are listed on their SkillMD profile.

Bio Long Read Sequencing Nanopore Methylation by majiayu000

Name: Bio Long Read Sequencing Nanopore Methylation
Rating: 4.2 (5 reviews)
Author: majiayu000

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.

majiayu000 89a2ea8 2 files · 3.0 KB Updated Sep 9, 2026 567 repo stars

File contents

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 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

---name: bio-long-read-sequencing-nanopore-methylationdescription: 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.--- # Nanopore Methylation Calling ## Modern Workflow (modkit) ONT's modkit is the recommended tool for methylation analysis from basecalled data. ### Extract Methylation from BAM ```bash # 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 ```bash # 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 ```bash # 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 ```bash # Get modification summary statistics modkit summary aligned.bam # Output includes: # - Total reads with modifications # - Modification types detected # - Fraction modified per type ``` ## Differential Methylation ```bash # 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

Frequently asked questions