Introduction

nf-core/demultiplex-demultiplex is a bioinformatics best-practice analysis pipeline to optionally demultiplex fastq files run bismark against a human reference and pUC19 methylated and unmethylated controls.

The pipeline is built using Nextflow, 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. The Nextflow DSL2 implementation of this pipeline uses one container per process which makes it much easier to maintain and update software dependencies. Where possible, these processes have been submitted to and installed from nf-core/modules in order to make them available to all nf-core pipelines, and to everyone within the Nextflow community!

Pipeline summary

This pipeline will process methylseq data including RRBS.

1. Demultiplex if necessary (Demultiplex)
2. Read QC (FastQC)
3. Present QC for raw reads (MultiQC)
4. Trim using RRBS specific flags with TrimGalore (Trim Galore)
5. Align to reference genome with BISMARK (bismark)
6. Align to fully methylated control with BISMARK (bismark)
7. Align to unmethylated control with BISMARK (bismark)
8. Bismark methylation extractor (bismark)
9. optional: filter Bismark methylation extractor output using a bed file
10. Bisulphite conversion assessment
11. Post alignment QC

slurm specific instructions

1. Download the pipeline:

   git clone https://github.com/chelauk/nf-core-demultiplex-methylation.git

2. Edit your .bashrc file to set the following variables:

   # Set all the Singularity cache dirs to Scratch
   export SINGULARITY_CACHEDIR=**/your/selected/scratch/folder/singularity_imgs**
   export SINGULARITY_TMPDIR=$SINGULARITY_CACHEDIR/tmp
   export SINGULARITY_LOCALCACHEDIR=$SINGULARITY_CACHEDIR/localcache
   export SINGULARITY_PULLFOLDER=$SINGULARITY_CACHEDIR/pull
   # match the NXF_SINGULARITY_CACHEDIR
   export NXF_SINGULARITY_CACHEDIR=**/your/selected/scratch/folder/singularity_imgs**

3. Start running your own analysis edit a sbatch script

   #!/bin/bash -l
   #SBATCH --job-name=demultiplex
   #SBATCH --output=nextflow_out.txt
   #SBATCH --partition=master-worker
   #SBATCH --ntasks=1
   #SBATCH --time=120:00:00
   
   nextflow run **/location/of/your/nextflow_pipelines/nf-core-demultiplex-methylation** \
   --input input.csv  \
   -profile slurm,singularity \
   -resume

4. Start your sbatch job:

   sbatch runNextflow.sh

Documentation

The nf-core/demultiplex pipeline comes with documentation about the pipeline usage and output.

Credits

nf-core-demultiplex-methylation was originally written by Chela James

We thank the following people for their extensive assistance in the development of this pipeline:

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

Citations

An extensive list of references for the tools used by the pipeline can be found in the 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.