nf-core pipeline · bisulfite-sequencing, dna-methylation, em-seq
nf-core/methylseq
A Nextflow pipeline for quality control, alignment and methylation extraction from bisulfite and TAPS sequencing data.
What nf-core/methylseq is
nf-core/methylseq is an analysis pipeline designed for processing DNA methylation sequencing data, including bisulfite sequencing and TAPS (TET-assisted pyridine borane sequencing). It carries out data pre-processing from raw FastQ inputs, performs read alignment, handles duplicate marking, extracts methylation calls, and executes quality-control evaluations across all samples.
Originally authored by Phil Ewels and maintained by Sateesh Peri, the pipeline was developed within the nf-core community. It offers interchangeable sub-workflows, enabling researchers to choose between established alignment and methylation extraction suites such as Bismark, bwa-meth with MethylDackel, or BWA-Mem combined with rastair.
Built using the Nextflow workflow engine, nf-core/methylseq provides reproducible data processing through container runtimes including Docker, Singularity, Podman, Charliecloud, and Apptainer. It also supports GPU-accelerated alignment steps via NVIDIA Parabricks implementations.
What the pipeline does
The workflow begins by merging any re-sequenced FastQ files using cat, followed by initial read quality control using FastQC and adapter trimming via Trim Galore!. Read alignment can then follow several distinct paths based on user configuration: Bismark (utilising Bowtie 2 or HISAT2), bwa-meth (with optional BWA-MEM2 acceleration via the --use_mem2 flag), or BWA-Mem. Where GPU resources and container engines are present, Parabricks GPU implementations (fq2bammeth or fq2bammem) can be deployed instead.
Following alignment, duplicate reads are removed or flagged using Bismark or Picard MarkDuplicates. Cytosine methylation calls are subsequently extracted using Bismark, MethylDackel, or the rastair sub-workflow for TAPS data. Finally, post-alignment assessment is performed with optional Qualimap and Preseq evaluations, culminating in aggregated quality-control reporting via MultiQC.
Key facts
- Pipeline engine
- Nextflow
- Latest release
- 4.2.0 (2025-12-12)
- Original author
- Phil Ewels
- Current maintainer
- Sateesh Peri
- Input formats
- FastQ (single-end or paired-end) via samplesheet CSV
- Primary aligners
- Bismark (Bowtie 2 / HISAT2), bwa-meth, BWA-Mem, and NVIDIA Parabricks
- Methylation callers
- Bismark, MethylDackel, rastair
- Quality control tools
- FastQC, Trim Galore!, Qualimap, Preseq, MultiQC
- Container support
- Docker, Singularity, Podman, Charliecloud, Apptainer
What you can use it for
- Bisulfite sequencing methylation analysis
- Align bisulfite-treated single-end or paired-end sequencing reads and extract cytosine methylation levels across the genome using Bismark or bwa-meth combined with MethylDackel.
- TAPS data processing
- Process TET-assisted pyridine borane sequencing reads by aligning with BWA-Mem or bwa-meth and resolving methylation calls using rastair.
- Targeted methylation profiling
- Restrict analysis and alignment evaluations to predefined genomic loci using the targeted sequencing workflow with target regions files.
- GPU-accelerated bisulfite alignment
- Accelerate read alignment workflows on GPU infrastructure using Parabricks implementations of bwa-meth and BWA-Mem.
Run it here
The research agent runs nf-core/methylseq with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
Strengths and limits
Good at
- Supports multiple dedicated methylation alignment and calling workflows, including Bismark, bwa-meth, and BWA-Mem with rastair.
- Accommodates GPU acceleration for bwa-meth and BWA-Mem alignments via NVIDIA Parabricks container profiles.
- Performs end-to-end quality control and reporting by integrating FastQC, Trim Galore!, Qualimap, Preseq, and MultiQC.
- Provides portable and reproducible execution across diverse environments using container platforms including Docker, Apptainer, and Singularity.
Watch out for
- GPU acceleration via Parabricks requires container runtimes (Docker, Singularity, or Podman) and is not supported using Conda or Mamba.
- Sample reports and overall summary reports are generated natively within the Bismark workflow but are not provided by the bwa-meth or rastair pathways.
Questions
Can I run nf-core/methylseq without my own cluster?
The research agent runs nf-core/methylseq with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
What licence is nf-core/methylseq under?
MIT, like every nf-core pipeline.
Which aligners are available in the pipeline?
Users can select between Bismark using Bowtie 2 (--aligner bismark), Bismark using HISAT2 (--aligner bismark_hisat), bwa-meth (--aligner bwameth), and BWA-Mem (--aligner bwamem).
How is TAPS data processed in nf-core/methylseq?
TAPS data can be analysed using BWA-Mem coupled with rastair, or alternatively by using bwa-meth with the --taps parameter flag.
Can the pipeline process targeted methylation sequencing data?
Yes, targeted sequencing analysis is supported by supplying the --run_targeted_sequencing flag alongside a regions definition via --target_regions_file.
What inputs does the pipeline require?
The pipeline requires a CSV samplesheet specifying sample identifiers and paths to single-end or paired-end FastQ files, along with an appropriate reference genome.
How to cite it
Ewels, P. et al. nf-core/methylseq: 10.5281/zenodo.1343417; Ewels, P., Peltzer, A., Fillinger, S., Patel, H., Alneberg, J., Wilm, A., Garcia, M. U., Di Tommaso, P. & Nahnsen, S. The nf-core framework for community-curated bioinformatics pipelines. Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.
Written from nf-core/methylseq's own README. Statements citing a figure that does not appear there are removed before publication. Licence and running details come from our registry.