# nf-core/methylseq

#

[![GitHub Actions CI Status](https://github.com/nf-core/methylseq/actions/workflows/nf-test.yml/badge.svg)](https://github.com/nf-core/methylseq/actions/workflows/nf-test.yml) [![GitHub Actions Linting Status](https://github.com/nf-core/methylseq/actions/workflows/linting.yml/badge.svg)](https://github.com/nf-core/methylseq/actions/workflows/linting.yml)[![AWS CI](https://img.shields.io/badge/CI%20tests-full%20size-FF9900?labelColor=000000&logo=Amazon%20AWS)](https://nf-co.re/methylseq/results)[![Cite with Zenodo](http://img.shields.io/badge/DOI-10.5281/zenodo.1343417-1073c8?labelColor=000000)](https://doi.org/10.5281/zenodo.1343417) [![nf-test](https://img.shields.io/badge/unit_tests-nf--test-337ab7.svg)](https://www.nf-test.com) [![Nextflow](https://img.shields.io/badge/version-%E2%89%A524.10.5-green?style=flat&logo=nextflow&logoColor=white&color=%230DC09D&link=https%3A%2F%2Fnextflow.io)](https://www.nextflow.io/) [![nf-core template version](https://img.shields.io/badge/nf--core_template-3.3.2-green?style=flat&logo=nfcore&logoColor=white&color=%2324B064&link=https%3A%2F%2Fnf-co.re)](https://github.com/nf-core/tools/releases/tag/3.3.2) [![run with conda](http://img.shields.io/badge/run%20with-conda-3EB049?labelColor=000000&logo=anaconda)](https://docs.conda.io/en/latest/) [![run with docker](https://img.shields.io/badge/run%20with-docker-0db7ed?labelColor=000000&logo=docker)](https://www.docker.com/) [![run with singularity](https://img.shields.io/badge/run%20with-singularity-1d355c.svg?labelColor=000000)](https://sylabs.io/docs/) [![Launch on Seqera Platform](https://img.shields.io/badge/Launch%20%F0%9F%9A%80-Seqera%20Platform-%234256e7)](https://cloud.seqera.io/launch?pipeline=https://github.com/nf-core/methylseq) [![Get help on Slack](http://img.shields.io/badge/slack-nf--core%20%23methylseq-4A154B?labelColor=000000&logo=slack)](https://nfcore.slack.com/channels/methylseq)[![Follow on Bluesky](https://img.shields.io/badge/bluesky-%40nf__core-1185fe?labelColor=000000&logo=bluesky)](https://bsky.app/profile/nf-co.re)[![Follow on Mastodon](https://img.shields.io/badge/mastodon-nf__core-6364ff?labelColor=FFFFFF&logo=mastodon)](https://mstdn.science/@nf_core)[![Watch on YouTube](http://img.shields.io/badge/youtube-nf--core-FF0000?labelColor=000000&logo=youtube)](https://www.youtube.com/c/nf-core) ## Introduction **nf-core/methylseq** is a bioinformatics analysis pipeline used for Methylation (Bisulfite) sequencing data. It pre-processes raw data from FastQ inputs, aligns the reads and performs extensive quality-control on the results. ![nf-core/methylseq metro map](docs/images/4.0.0_metromap.png) The pipeline is built using [Nextflow](https://www.nextflow.io), a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It uses Docker / Singularity / Podman / Charliecloud / Apptainer containers making installation trivial and results highly reproducible. On release, automated continuous integration tests run the pipeline on a full-sized dataset on the AWS cloud infrastructure. This ensures that the pipeline runs on AWS, has sensible resource allocation defaults set to run on real-world datasets, and permits the persistent storage of results to benchmark between pipeline releases and other analysis sources.The results obtained from the full-sized test can be viewed on the [nf-core website](https://nf-co.re/methylseq/results). > Read more about **Bisulfite Sequencing & Three-Base Aligners** used in this pipeline [here](docs/usage/bs-seq-primer.md) ## Pipeline Summary The pipeline allows you to choose between running either [Bismark](https://github.com/FelixKrueger/Bismark) or [bwa-meth](https://github.com/brentp/bwa-meth) / [MethylDackel](https://github.com/dpryan79/methyldackel). Choose between workflows by using `--aligner bismark` (default, uses bowtie2 for alignment), `--aligner bismark_hisat` or `--aligner bwameth`. For higher performance, the pipeline can leverage the [Parabricks implementation of bwa-meth (fq2bammeth)](https://docs.nvidia.com/clara/parabricks/latest/documentation/tooldocs/man_fq2bam_meth.html), which implements the baseline tool `bwa-meth` in a performant method using fq2bam (BWA-MEM + GATK) as a backend for processing on GPU. To use this option, include the `gpu` profile along with `--aligner bwameth`. Note: For faster CPU runs with BWA-Meth, enable the BWA-MEM2 algorithm using `--use_mem2`. The GPU pathway (Parabricks) requires `-profile gpu` and a container runtime (Docker, Singularity, or Podman); Conda/Mamba are not supported for the GPU module. | Step | Bismark workflow | bwa-meth workflow | | -------------------------------------------- | ------------------------ | --------------------- | | Generate Reference Genome Index _(optional)_ | Bismark | bwa-meth | | Merge re-sequenced FastQ files | cat | cat | | Raw data QC | FastQC | FastQC | | Adapter sequence trimming | Trim Galore! | Trim Galore! | | Align Reads | Bismark (bowtie2/hisat2) | bwa-meth | | Deduplicate Alignments | Bismark | Picard MarkDuplicates | | Extract methylation calls | Bismark | MethylDackel | | Sample report | Bismark | - | | Summary Report | Bismark | - | | Alignment QC | Qualimap _(optional)_ | Qualimap _(optional)_ | | Sample complexity | Preseq _(optional)_ | Preseq _(optional)_ | | Project Report | MultiQC | MultiQC | Optional targeted sequencing analysis is available via `--run_targeted_sequencing` and `--target_regions_file`; see the [usage documentation](https://nf-co.re/methylseq/usage) for details. ## Usage > [!NOTE] > If you are new to Nextflow and nf-core, please refer to [this page](https://nf-co.re/docs/usage/installation) on how to set-up Nextflow. Make sure to [test your setup](https://nf-co.re/docs/usage/introduction#how-to-run-a-pipeline) with `-profile test` before running the workflow on actual data. First, prepare a samplesheet with your input data that looks as follows: `samplesheet.csv`: ```csv sample,fastq_1,fastq_2,genome SRR389222_sub1,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub1.fastq.gz,, SRR389222_sub2,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub2.fastq.gz,, SRR389222_sub3,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub3.fastq.gz,, Ecoli_10K_methylated,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/Ecoli_10K_methylated_R1.fastq.gz,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/Ecoli_10K_methylated_R2.fastq.gz, ``` > Each row represents a fastq file (single-end) or a pair of fastq files (paired end). Now, you can run the pipeline using default parameters as: ```bash nextflow run nf-core/methylseq --input samplesheet.csv --outdir --genome GRCh37 -profile ``` > [!WARNING] > Please provide pipeline parameters via the CLI or Nextflow `-params-file` option. Custom config files including those provided by the `-c` Nextflow option can be used to provide any configuration _**except for parameters**_; see [docs](https://nf-co.re/docs/usage/getting_started/configuration#custom-configuration-files). For more details and further functionality, please refer to the [usage documentation](https://nf-co.re/methylseq/usage) and the [parameter documentation](https://nf-co.re/methylseq/parameters). ## Pipeline output To see the results of an example test run with a full size dataset refer to the [results](https://nf-co.re/methylseq/results) tab on the nf-core website pipeline page. For more details about the output files and reports, please refer to the [output documentation](https://nf-co.re/methylseq/output). ## Credits nf-core/methylseq was originally written by Phil Ewels ([@ewels](https://github.com/ewels)), and Sateesh Peri ([@sateeshperi](https://github.com/sateeshperi)) is its active maintainer. We thank the following people for their extensive assistance in the development of this pipeline: - Felix Krueger ([@FelixKrueger](https://github.com/FelixKrueger)) - Edmund Miller ([@EMiller88](https://github.com/emiller88)) - Rickard Hammarén ([@Hammarn](https://github.com/Hammarn/)) - Alexander Peltzer ([@apeltzer](https://github.com/apeltzer/)) - Patrick Hüther ([@phue](https://github.com/phue/)) - Maxime U Garcia ([@maxulysse](https://github.com/maxulysse/)) ## Contributions and Support If you would like to contribute to this pipeline, please see the [contributing guidelines](.github/CONTRIBUTING.md). For further information or help, don't hesitate to get in touch on the [Slack `#methylseq` channel](https://nfcore.slack.com/channels/methylseq) (you can join with [this invite](https://nf-co.re/join/slack)). ## Citations If you use nf-core/methylseq for your analysis, please cite it using the following doi: [10.5281/zenodo.1343417](https://doi.org/10.5281/zenodo.1343417) An extensive list of references for the tools used by the pipeline can be found in the [`CITATIONS.md`](CITATIONS.md) file. You can cite the `nf-core` publication as follows: > **The nf-core framework for community-curated bioinformatics pipelines.** > > Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen. > > _Nat Biotechnol._ 2020 Feb 13. doi: [10.1038/s41587-020-0439-x](https://dx.doi.org/10.1038/s41587-020-0439-x).

License
  MIT

## Contents

- ["nf-core/methylseq"]
- assets/
- conf/
- docs/
- docs/images/
- modules/
- modules/nf-core/
- workflows/
- subworkflows/
- nextflow.config
- README.md
- nextflow_schema.json
- CHANGELOG.md
- LICENSE
- CODE_OF_CONDUCT.md
- CITATIONS.md
- modules.json
- docs/usage.md
- docs/output.md
- .nf-core.yml
- .pre-commit-config.yaml
- .prettierignore