Methylation pipeline

Version Compatibility

Reference examples tested with: Bismark 0.24+, Bowtie2 2.5.3+, FastQC 0.12+, Trim Galore 0.6.10+, fastp 0.23+, methylKit 1.28+

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

• R: packageVersion('<pkg>') then ?function_name to verify parameters
• 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.

Methylation Pipeline

"Analyze my bisulfite sequencing data from FASTQ to DMRs" -> Chain QC/trim, Bismark alignment + dedup, methylation calling, coverage-filtered per-CpG testing, and selection-aware DMR detection.

• CLI + R: Trim Galore/fastp -> bismark -> deduplicate_bismark -> bismark_methylation_extractor -> methylKit (filter/normalize/unite) -> DSS/dmrseq

This is a workflow skill: it owns the chaining decisions and hand-offs, not the internals of any one step. Every step below cross-references the component skill that teaches its mechanism.

The governing principle

A methylation callset is decided at four seams, not inside the caller.

1. Bisulfite conversion is verified BEFORE any beta value is trusted — this is the gate that most pipelines skip. An unmethylated lambda (or spike-in) bounds UNDER-conversion (residual C read as methylated -> false hyper); a methylated pUC19 control bounds OVER-conversion (5mC deaminated -> false hypo). Require conversion >99% from the lambda control before computing a single methylation level; a 98% library silently shifts every call.
2. Mate overlap must be deduplicated once. In paired-end WGBS the R1/R2 insert overlaps, and counting a CpG in both mates double-weights it. bismark_methylation_extractor --paired-end applies --no_overlap by default; running the extractor in single-end mode on paired data (or losing --no_overlap) inflates coverage and distorts levels.
3. M-bias is trimmed from the plot, and coverage is filtered BEFORE testing. End-repair fill-in biases the first/last few bases (read the M-bias plot, trim positionally — not a fixed number). Then filter low- and extreme-coverage CpGs before any test: variance depends on coverage, so unfiltered low-coverage sites dominate the FDR.
4. The statistic must respect counts, and region FDR must respect selection. A bare-beta t-test discards coverage (the precision unique to sequencing); use a beta-binomial/overdispersion model (DSS, methylKit overdispersion='MN') for counts, or limma-on-M-values for arrays. For REGIONS, methylKit fixed tiles do not correct for the region-selection step — use dmrseq (permutation null over selection) or DSS callDMR for a rigorous region-level FDR.

Made-once commitments

Commitment	Choice	Consequence inherited downstream
Genome build + library model	One build; directional (WGBS/EM-seq) vs non-directional/PBAT	Wrong strand model tanks mapping; build fixes all coordinates
Conversion controls	Lambda (unmethylated) + pUC19 (methylated) spike-ins	Without them, under/over-conversion is undetectable and biases every call
Assay entry	WGBS/EM-seq (this pipeline) vs Infinium array (array-preprocessing)	Array data enters at beta/M matrix, not Bismark
Context	CpG (default) vs CHG/CHH (plants/non-CpG)	Non-CpG contexts need conversion-aware calling and separate testing

Workflow Overview

FASTQ files
    |
    v
[1. QC & Trimming] -----> fastp/Trim Galore
    |
    v
[2. Alignment] ---------> Bismark
    |
    v
[3. Deduplication] -----> deduplicate_bismark
    |
    v
[4. Methylation Calling] -> bismark_methylation_extractor
    |
    v
[5. Per-CpG Analysis] ---> methylKit (R) or scipy (Python)
    |
    v
[6. DMR Detection] ------> methylKit/DSS
    |
    v
Differentially methylated regions

Primary Path: Bismark + methylKit

Step 1: Quality Control

# Trim Galore recommended for bisulfite data (handles adapter bias)
trim_galore --paired --fastqc \
    -o trimmed/ \
    sample_R1.fastq.gz sample_R2.fastq.gz

# Or fastp with conservative settings
fastp -i sample_R1.fastq.gz -I sample_R2.fastq.gz \
    -o trimmed/sample_R1.fq.gz -O trimmed/sample_R2.fq.gz \
    --detect_adapter_for_pe \
    --qualified_quality_phred 20 \
    --length_required 35 \
    --html qc/sample_fastp.html

Step 2: Bismark Alignment

# Prepare genome (once)
bismark_genome_preparation --bowtie2 genome/

# Align
bismark --genome genome/ \
    -1 trimmed/sample_R1_val_1.fq.gz \
    -2 trimmed/sample_R2_val_2.fq.gz \
    -o aligned/ \
    --parallel 4 \
    --temp_dir tmp/

# Output: sample_R1_val_1_bismark_bt2_pe.bam

QC Checkpoint: Check Bismark report

• Mapping efficiency >50% (the 3-letter alphabet lowers uniqueness; 50-70% is normal for WGBS)
• Bisulfite conversion >99% from the unmethylated lambda spike-in (bounds under-conversion -> false hyper); also check a methylated pUC19 control for over-conversion (-> false hypo). With NO spike-in, use the genome-wide non-CpG (CHH) methylation rate as a conversion proxy in mammals (expected near 0)

Step 3: Deduplication (WGBS / EM-seq ONLY)

Deduplicate WGBS and EM-seq. Do NOT deduplicate RRBS, amplicon, or other target-enrichment libraries: their reads legitimately stack at the MspI cut sites, so positional dedup destroys real coverage (Bismark's own docs say so). Skip this step entirely for RRBS.

# WGBS / EM-seq only -- skip for RRBS/amplicon
deduplicate_bismark \
    --bam \
    -p \
    --output_dir deduplicated/ \
    aligned/sample_R1_val_1_bismark_bt2_pe.bam

Step 4: Methylation Calling

# --paired-end enables --no_overlap by DEFAULT (deduplicates the R1/R2 insert overlap so a CpG in
# the overlap is not double-counted). Do NOT run the extractor in single-end mode on paired data.
bismark_methylation_extractor \
    --paired-end \
    --comprehensive \
    --bedGraph \
    --cytosine_report \
    --genome_folder genome/ \
    -o methylation/ \
    deduplicated/sample_R1_val_1_bismark_bt2_pe.deduplicated.bam

# Generate summary report
bismark2report
bismark2summary

Step 5: Analysis with methylKit

Goal: Turn per-sample coverage/cytosine reports into a coverage-filtered, normalized, united methylation object ready for testing.

Approach: Read each sample with the matching pipeline, drop low-coverage and extreme-coverage CpGs, normalize coverage across libraries, then unite to the sites covered in every sample.

library(methylKit)

# Read methylation calls
files <- list(
    'methylation/control_1.CpG_report.txt',
    'methylation/control_2.CpG_report.txt',
    'methylation/treated_1.CpG_report.txt',
    'methylation/treated_2.CpG_report.txt'
)

sample_ids <- c('control_1', 'control_2', 'treated_1', 'treated_2')
treatment <- c(0, 0, 1, 1)

# Read cytosine reports
meth_obj <- methRead(
    location = as.list(files),
    sample.id = as.list(sample_ids),
    assembly = 'hg38',
    treatment = treatment,
    context = 'CpG',
    pipeline = 'bismarkCytosineReport'
)

# Filter by coverage
meth_filtered <- filterByCoverage(meth_obj, lo.count = 10, hi.perc = 99.9)

# Normalize coverage
meth_norm <- normalizeCoverage(meth_filtered)

# Merge samples (keep sites covered in all)
meth_merged <- unite(meth_norm, destrand = TRUE)

# Sample statistics
getMethylationStats(meth_obj[[1]], plot = TRUE)
getCoverageStats(meth_obj[[1]], plot = TRUE)

Step 5b: Python Alternative for Per-CpG Testing

When methylKit is unavailable or a Python-only workflow is preferred, per-CpG testing can be performed with scipy and statsmodels on beta values computed from the coverage files.

import pandas as pd
from scipy.stats import ttest_ind
from statsmodels.stats.multitest import multipletests
import numpy as np

# Read Bismark coverage files and compute beta values
# beta = count_methylated / (count_methylated + count_unmethylated)
# Filter CpGs with < 10x coverage in any sample
# Run per-CpG Welch's t-test between groups
# Apply BH FDR correction: multipletests(pvals, method='fdr_bh')
# See methylation-analysis/differential-cpg-testing for full pipeline

A bare-beta t-test discards coverage (the precision information unique to sequencing) and is only a quick look. For sequencing counts, route to a beta-binomial / overdispersion-corrected count model (DSS, or methylKit with overdispersion='MN'); for array or continuous data, use limma on M-values. The count-vs-continuous decision is owned by methylation-analysis/differential-cpg-testing.

Step 6: DMR Detection

methylKit fixed tiles are a fast screen, but their region q-value is not corrected for the region-selection step. For a rigorous region-level FDR use dmrseq (a permutation null over the selection) or DSS callDMR, and confirm with cross-tool overlap - see methylation-analysis/dmr-detection.

# Calculate differential methylation (per CpG). overdispersion='MN' + test='Chisq' applies the
# overdispersion correction seam #4 requires; the default 'none' gives underdispersed p-values.
diff_meth <- calculateDiffMeth(meth_merged, overdispersion = 'MN', test = 'Chisq')

# Get significant DMCs
dmc <- getMethylDiff(diff_meth, difference = 25, qvalue = 0.01)

# Tile into regions (DMRs)
tiles <- tileMethylCounts(meth_merged, win.size = 1000, step.size = 1000)
diff_tiles <- calculateDiffMeth(tiles, overdispersion = 'MN', test = 'Chisq')   # same overdispersion correction as per-CpG (seam #4)
dmr <- getMethylDiff(diff_tiles, difference = 25, qvalue = 0.01)

# Export
write.csv(as.data.frame(dmc), 'dmc_results.csv')
write.csv(as.data.frame(dmr), 'dmr_results.csv')

# Annotate with genomic features
library(genomation)
gene_obj <- readTranscriptFeatures('genes.bed')
annotateWithGeneParts(as(dmr, 'GRanges'), gene_obj)

Parameter Recommendations

Step	Parameter	Value
Trim Galore	default	Recommended for BS-seq
Bismark	--parallel	4 (per sample parallelization)
methylKit	lo.count	10 (minimum coverage)
methylKit	difference	25 (% methylation difference)
methylKit	qvalue	0.01
DMR tiles	win.size	500-1000 bp

Common Errors

Symptom	Cause	Fix
Genome-wide hyper- or hypo-methylation shift	Under/over-conversion never checked	Gate on lambda (>99%) + pUC19 controls BEFORE trusting any beta value
Coverage inflated, levels off in mate-overlap regions	Extractor run single-end on paired data / lost --no_overlap	Use --paired-end (applies --no_overlap); do not single-end paired data
Systematic bias at read ends	M-bias from end-repair fill-in	Trim positionally from the M-bias plot, not a fixed number
Low-coverage CpGs dominate the DMC list	No coverage filter before testing	filterByCoverage(lo.count=10, hi.perc=99.9) before calculateDiffMeth
Spurious DMCs / underdispersed p-values	Bare-beta t-test ignores counts/overdispersion	Beta-binomial/DSS or methylKit overdispersion='MN'; limma-M for arrays
Region q-values too optimistic	methylKit fixed tiles ignore the region-selection step	Use dmrseq (permutation null) or DSS callDMR for region-level FDR
Very low mapping efficiency	Wrong library directionality (PBAT/non-directional aligned as directional)	Set the correct Bismark strand model (methylation-analysis/bismark-alignment)

The full per-step chain is shown above; the runnable methylKit analysis is in this skill's examples/ (methylkit_analysis.R).

References

• Krueger F, Andrews SR (2011) Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27:1571-1572. DOI 10.1093/bioinformatics/btr167.
• Akalin A, Kormaksson M, Li S, et al (2012) methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biology 13:R87. DOI 10.1186/gb-2012-13-10-r87.
• Feng H, Conneely KN, Wu H (2014) A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data. Nucleic Acids Research 42:e69. DOI 10.1093/nar/gku154. (DSS.)
• Korthauer K, Chakraborty S, Benjamini Y, Irizarry RA (2019) Detection and accurate false discovery rate control of differentially methylated regions from whole genome bisulfite sequencing. Biostatistics 20:367-383. DOI 10.1093/biostatistics/kxy007. (dmrseq; region-selection-aware FDR.)

Related Skills

• methylation-analysis/bismark-alignment - Bisulfite/EM-seq alignment, library/strand model, conversion QC
• methylation-analysis/methylation-calling - Calling from BAM (Bismark/MethylDackel), contexts, variant-aware
• methylation-analysis/methylkit-analysis - methylKit object model and overdispersion gotchas
• methylation-analysis/differential-cpg-testing - Per-CpG testing (count-vs-continuous fork)
• methylation-analysis/dmr-detection - Selection-aware region callers (dmrseq/DSS) and PMD segmentation
• methylation-analysis/array-preprocessing - Alternate entry: Infinium IDAT to beta/M matrix
• methylation-analysis/cell-type-deconvolution - Cell-fraction covariates for bulk-tissue EWAS
• methylation-analysis/epigenetic-clocks - DNAm age and age acceleration
• methylation-analysis/ewas-design - EWAS confounding, batch, inflation, and replication