Bio Workflows Methylation Pipeline

---
name: bio-workflows-methylation-pipeline
description: End-to-end bisulfite sequencing workflow from FASTQ to differentially methylated regions. Covers Bismark alignment, methylation calling, and DMR detection with methylKit. Use when analyzing bisulfite sequencing data.
tool_type: mixed
primary_tool: Bismark
workflow: true
depends_on:
  - read-qc/fastp-workflow
  - methylation-analysis/bismark-alignment
  - methylation-analysis/methylation-calling
  - methylation-analysis/methylkit-analysis
  - methylation-analysis/differential-cpg-testing
  - methylation-analysis/dmr-detection
qc_checkpoints:
  - after_qc: "Q30 >80%, adapter content removed"
  - after_alignment: "Mapping efficiency >50%, bisulfite conversion >99%"
  - after_calling: "Coverage distribution reasonable, no biased positions"
---

## 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"** -> Orchestrate Bismark alignment, methylation calling, methylKit analysis, DMR detection, annotation with genomic features, and visualization of methylation patterns.

Complete workflow from bisulfite sequencing FASTQ to differentially methylated regions.

## 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

```bash
# 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

```bash
# 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)

### Step 3: Deduplication

```bash
deduplicate_bismark \
    --bam \
    -p \
    -o deduplicated/ \
    aligned/sample_R1_val_1_bismark_bt2_pe.bam
```

### Step 4: Methylation Calling

```bash
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.

```r
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.

```python
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.

```r
# Calculate differential methylation (per CpG)
diff_meth <- calculateDiffMeth(meth_merged)

# 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)
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 |

## Troubleshooting

| Issue | Likely Cause | Solution |
|-------|--------------|----------|
| Low mapping rate | Normal for BS-seq | Expect 40-70% |
| Low conversion | Failed bisulfite treatment | Check spike-in controls |
| Few DMRs | Low coverage, small differences | Increase sequencing, relax thresholds |
| Biased positions | M-bias (end-repair fill-in) | Trim the affected read ends from the M-bias plot, not a fixed number |

## Complete Pipeline Script

```bash
#!/bin/bash
set -e

THREADS=4
GENOME="genome/"
SAMPLES="control_1 control_2 treated_1 treated_2"
OUTDIR="methylation_results"

mkdir -p ${OUTDIR}/{trimmed,aligned,deduplicated,methylation,qc}

# Step 1: QC
for sample in $SAMPLES; do
    trim_galore --paired --fastqc -o ${OUTDIR}/trimmed/ \
        ${sample}_R1.fastq.gz ${sample}_R2.fastq.gz
done

# Step 2: Alignment
for sample in $SAMPLES; do
    bismark --genome ${GENOME} \
        -1 ${OUTDIR}/trimmed/${sample}_R1_val_1.fq.gz \
        -2 ${OUTDIR}/trimmed/${sample}_R2_val_2.fq.gz \
        -o ${OUTDIR}/aligned/ \
        --parallel ${THREADS} --temp_dir tmp/
done

# Step 3: Deduplication
for sample in $SAMPLES; do
    deduplicate_bismark --bam -p \
        -o ${OUTDIR}/deduplicated/ \
        ${OUTDIR}/aligned/${sample}_R1_val_1_bismark_bt2_pe.bam
done

# Step 4: Methylation calling
for sample in $SAMPLES; do
    bismark_methylation_extractor --paired-end --comprehensive \
        --bedGraph --cytosine_report \
        --genome_folder ${GENOME} \
        -o ${OUTDIR}/methylation/ \
        ${OUTDIR}/deduplicated/${sample}_R1_val_1_bismark_bt2_pe.deduplicated.bam
done

bismark2report
echo "Pipeline complete. Run R script for DMR analysis."
```

## 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