Analyze DNA methylation in Oxford Nanopore data without bisulfite conversion. Use modkit to summarize modified-base information into per-site methylation frequencies, inspect sample statistics, and focus analysis on CpG islands or promoter regions.
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.
Run score Not tested
See how to installReview the instructions the agent will receive before you install.
Reference examples tested with: methylKit 1.28+, minimap2 2.26+, samtools 1.19+
Before using code patterns, verify installed versions match. If versions differ:
<tool> --version then <tool> --help to confirm flagsIf code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.
"Call methylation from my Nanopore reads" → Extract 5mC/6mA modification probabilities from basecalled reads and summarize per-site methylation frequencies.
modkit pileup aligned.bam methylation.bed --ref ref.faONT's modkit is the recommended tool for methylation analysis from basecalled data.
# Assumes BAM has MM/ML tags from dorado basecalling
modkit pileup input.bam methylation.bed \
--ref reference.fa \
--cpg \
--combine-strands
# 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
# 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
# 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
# Get modification summary statistics
modkit summary aligned.bam
# Output includes:
# - Total reads with modifications
# - Modification types detected
# - Fraction modified per type
# 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
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