Epigenomics • Methylation
Authority: Babraham Bioinformatics / nf-core/methylseq
Production Blueprint v4.59. Whole-Genome Bisulfite Sequencing (WGBS) Methylation
Base-resolution cytosine methylation detection across CpG, CHG, and CHH contexts, distinguishing 5-methylcytosine from unmethylated cytosines using 3-letter in silico bisulfite alignments.
| Metric / Requirement | Specification | Target Configuration |
|---|---|---|
| Core Engine / Framework | Bismark / bwa-meth + methylKit | Nextflow / Snakemake Compatible |
| Benchmark Runtime | ~12 – 16 hours (30× Human WGBS) | 32 CPU cores / 64 GB RAM |
| Standard Container | quay.io/biocontainers/bismark:0.24.2--hdfd78af_0 | Docker • Singularity • Conda |
| Input → Deliverable | Bisulfite FASTQ → Bismark BAM, Methylation Call Dictionaries, DMRs | |
Workflow Chain: Bisulfite FASTQ → Trim Galore → Bismark / bwa-meth → deduplicate_bismark → bismark_methylation_extractor → methylKit DMR analysis
⚡ Jump to Step:
Step 1 (Trim) ↓
Step 2 (Bismark) ↓
Step 3 (deduplicate_bismark) ↓
Step 4 (bismark_methylation_extractor) ↓
⚡
Step 1: Bisulfite Trimming & M-Bias Removal with Trim Galore
Trim Galore
Trims Illumina adapters and clips methylation bias (M-bias) introduced during end repair.
trim_galore --paired --fastqc --clip_r1 2 --clip_r2 2 \
--three_prime_clip_r1 2 --three_prime_clip_r2 2 \
sample_R1.fq.gz sample_R2.fq.gz
Key Flags & Notes:
--clip_r1 2 purges artificial unmethylated cytosines incorporated during end-repair.
⚡
Step 2: 3-Letter Bisulfite Reference Alignment with Bismark
Bismark v0.24+
Performs in silico C→T conversions on reads and reference to enable mapping without bisulfite penalty.
bismark --multicore 8 --genome /references/bismark_hg38/ \
-1 sample_R1_val_1.fq.gz -2 sample_R2_val_2.fq.gz \
-o bismark_out/
Key Flags & Notes:
--multicore 8 parallelizes across 4 parallel in silico reference strands.
⚡
Step 3: PCR Duplicate Marking for Bisulfite Reads
deduplicate_bismark
Removes duplicate read pairs while preserving strand-specific methylation orientations.
deduplicate_bismark --paired bismark_out/sample_R1_val_1_bismark_bt2_pe.bam
Key Flags & Notes: Specialized bisulfite deduplicator that avoids false collapsing of converted strands.
⚡
Step 4: Contextual Cytosine Methylation Extraction (CpG, CHG, CHH)
bismark_methylation_extractor
Decodes per-cytosine methylation ratios into bedGraph files across all sequence motifs.
bismark_methylation_extractor --paired-end --no_overlap \
--comprehensive --merge_non_CpG \
--bedGraph --counts --buffer_size 16G \
--cytosine_report --genome_folder /references/bismark_hg38/ \
sample_R1_val_1_bismark_bt2_pe.deduplicated.bam
Key Flags & Notes:
--no_overlap prevents double-counting cytosines on overlapping paired ends.🚀 Automated Nextflow (nf-core) Production Execution
Execute this complete workflow across any SLURM, AWS Batch, or Google Cloud HPC cluster using the verified community container pipeline:
nextflow run nf-core/methylseq -r 2.6.0 \
--input samplesheet.csv \
--genome GRCh38 \
--aligner bismark \
--outdir ./results_wgbs/ \
-profile docker