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## 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.
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 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/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 `--use_gpu` flag along with `--aligner bwameth`.
| 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 |
## 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).
