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"description": "<h1>\n <picture>\n <source media=\"(prefers-color-scheme: dark)\" srcset=\"docs/images/nf-core-methylseq_logo_dark.png\">\n <img alt=\"nf-core/methylseq\" src=\"docs/images/nf-core-methylseq_logo_light.png\">\n </picture>\n</h1>\n\n[](https://github.com/codespaces/new/nf-core/methylseq)\n[](https://github.com/nf-core/methylseq/actions/workflows/nf-test.yml)\n[](https://github.com/nf-core/methylseq/actions/workflows/linting.yml)[](https://nf-co.re/methylseq/results)[](https://doi.org/10.5281/zenodo.1343417)\n[](https://www.nf-test.com)\n\n[](https://www.nextflow.io/)\n[](https://github.com/nf-core/tools/releases/tag/3.5.1)\n[](https://docs.conda.io/en/latest/)\n[](https://www.docker.com/)\n[](https://sylabs.io/docs/)\n[](https://cloud.seqera.io/launch?pipeline=https://github.com/nf-core/methylseq)\n\n[](https://nfcore.slack.com/channels/methylseq)[](https://bsky.app/profile/nf-co.re)[](https://mstdn.science/@nf_core)[](https://www.youtube.com/c/nf-core)\n\n## Introduction\n\n**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.\n\n\n\nThe 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.\n\nOn 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).\n\n> Read more about **Bisulfite Sequencing & Three-Base Aligners** used in this pipeline [here](docs/usage/bs-seq-primer.md)\n\n## Pipeline Summary\n\nThe pipeline allows you to choose between running either [Bismark](https://github.com/FelixKrueger/Bismark), [bwa-meth](https://github.com/brentp/bwa-meth) / [MethylDackel](https://github.com/dpryan79/methyldackel) or [BWA-Mem](https://github.com/lh3/bwa) plus [rastair](https://bitbucket.org/bsblabludwig/rastair/src/master/) for TAPS data processing. rastair can also be used with bwa-meth aligned reads by setting the aligner to `--aligner bwameth` and adding the flag `--taps`.\n\nChoose between workflows by using `--aligner bismark` (default, uses bowtie2 for alignment), `--aligner bismark_hisat`, `--aligner bwameth` or `--aligner bwamem`. 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) and the [Parabricks implementation of bwa-mem (fq2bammemh)](https://docs.nvidia.com/clara/parabricks/latest/documentation/tooldocs/man_fq2bam.html), which implement the baseline tools `bwa-meth` and `bwa-mem`. To use this option, include the `gpu` profile along with `--aligner bwameth` or `--aligner bwamem`.\n\nNote: 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.\n\n| Step | Bismark workflow | bwa-meth workflow | bwa-mem + TAPS workflow |\n| -------------------------------------------- | ------------------------ | --------------------- | -------------------------- |\n| Generate Reference Genome Index _(optional)_ | Bismark | bwa-meth | bwa index |\n| Merge re-sequenced FastQ files | cat | cat | cat |\n| Raw data QC | FastQC | FastQC | FastQC |\n| Adapter sequence trimming | Trim Galore! | Trim Galore! | Trim Galore! |\n| Align Reads | Bismark (bowtie2/hisat2) | bwa-meth | bwa mem |\n| Deduplicate Alignments | Bismark | Picard MarkDuplicates | Picard MarkDuplicates |\n| Extract methylation calls | Bismark | MethylDackel | TAPS subworkflow (rastair) |\n| Sample report | Bismark | - | - |\n| Summary Report | Bismark | - | - |\n| Alignment QC | Qualimap _(optional)_ | Qualimap _(optional)_ | Qualimap _(optional)_ |\n| Sample complexity | Preseq _(optional)_ | Preseq _(optional)_ | Preseq _(optional)_ |\n| Project Report | MultiQC | MultiQC | MultiQC |\n\nOptional 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.\n\n## Usage\n\n> [!NOTE]\n> 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.\n\nFirst, prepare a samplesheet with your input data that looks as follows:\n\n`samplesheet.csv`:\n\n```csv\nsample,fastq_1,fastq_2,genome\nSRR389222_sub1,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub1.fastq.gz,,\nSRR389222_sub2,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub2.fastq.gz,,\nSRR389222_sub3,https://github.com/nf-core/test-datasets/raw/methylseq/testdata/SRR389222_sub3.fastq.gz,,\nEcoli_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,\n```\n\n> Each row represents a fastq file (single-end) or a pair of fastq files (paired end).\n\nNow, you can run the pipeline using default parameters as:\n\n```bash\nnextflow run nf-core/methylseq --input samplesheet.csv --outdir <OUTDIR> --genome GRCh37 -profile <docker/singularity/podman/shifter/charliecloud/conda/institute>\n```\n\n> [!WARNING]\n> 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).\n\nFor 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).\n\n## Pipeline output\n\nTo 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.\nFor more details about the output files and reports, please refer to the [output documentation](https://nf-co.re/methylseq/output).\n\n## Credits\n\nnf-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.\n\nWe thank the following people for their extensive assistance in the development of this pipeline:\n\n- Felix Krueger ([@FelixKrueger](https://github.com/FelixKrueger))\n- Edmund Miller ([@EMiller88](https://github.com/emiller88))\n- Rickard Hammar\u00e9n ([@Hammarn](https://github.com/Hammarn/))\n- Alexander Peltzer ([@apeltzer](https://github.com/apeltzer/))\n- Patrick H\u00fcther ([@phue](https://github.com/phue/))\n- Maxime U Garcia ([@maxulysse](https://github.com/maxulysse/))\n\n## Contributions and Support\n\nIf you would like to contribute to this pipeline, please see the [contributing guidelines](.github/CONTRIBUTING.md).\n\nFor 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)).\n\n## Citations\n\nIf 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)\n\nAn extensive list of references for the tools used by the pipeline can be found in the [`CITATIONS.md`](CITATIONS.md) file.\n\nYou can cite the `nf-core` publication as follows:\n\n> **The nf-core framework for community-curated bioinformatics pipelines.**\n>\n> Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.\n>\n> _Nat Biotechnol._ 2020 Feb 13. doi: [10.1038/s41587-020-0439-x](https://dx.doi.org/10.1038/s41587-020-0439-x).\n",
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