noirot/cutandrun

Analysis pipeline for CUT&RUN and CUT&TAG experiments that includes QC, support for spike-ins, IgG controls, peak calling and downstream analysis.

Introduction

nf-core/cutandrun is a best-practice bioinformatic analysis pipeline for CUT&Run and CUT&Tag experimental protocols that where developed to study protein-DNA interactions and epigenomic profiling.

The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It is capable of using 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 easier to maintain and update software dependencies. Where possible, these processes have been submitted to and installed from nf-core/modules.

The pipeline has been developed with continuous integration (CI) in mind. nf-core code and module linting as well as a battery of over 100 unit and integration tests run on pull request to the main repository and on release of the pipeline. On official release, automated CI 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.

Pipeline summary

1. Check input files
2. Merge re-sequenced FastQ files (cat)
3. Read QC (FastQC)
4. Adapter and quality trimming (Trim Galore!)
5. Alignment to both target and spike-in genomes (Bowtie 2)
6. Filter on quality, sort and index alignments (samtools)
7. Duplicate read marking (picard)
8. Create bedGraph files (bedtools
9. Create bigWig coverage files (bedGraphToBigWig)
10. Peak calling specifically tailored for low background noise experiments (SEACR)
11. Consensus peak merging and reporting (bedtools)
12. Quality control and analysis:
    1. Alignment, fragment length and peak analysis and replicate reproducibility (python)
    2. Heatmap peak analysis (deepTools)
13. Genome browser session (IGV)
14. Present QC for raw read, alignment and duplicate reads (MultiQC)

Quick Start

1. Install Nextflow (>=21.10.3)

2. Install any of Docker, Singularity, Podman, Shifter or Charliecloud for full pipeline reproducibility (please only use Conda as a last resort; see docs)

3. Download the pipeline and test it on a minimal dataset with a single command:

   nextflow run nf-core/cutandrun -profile test,YOURPROFILE

   Note that some form of configuration will be needed so that Nextflow knows how to fetch the required software. This is usually done in the form of a config profile (YOURPROFILE in the example command above). You can chain multiple config profiles in a comma-separated string.

   • The pipeline comes with config profiles called docker, singularity, podman, shifter, charliecloud and conda which instruct the pipeline to use the named tool for software management. For example, -profile test,docker.
   • Please check nf-core/configs to see if a custom config file to run nf-core pipelines already exists for your Institute. If so, you can simply use -profile <institute> in your command. This will enable either docker or singularity and set the appropriate execution settings for your local compute environment.
   • If you are using singularity and are persistently observing issues downloading Singularity images directly due to timeout or network issues, then you can use the --singularity_pull_docker_container parameter to pull and convert the Docker image instead. Alternatively, you can use the nf-core download command to download images first, before running the pipeline. Setting the NXF_SINGULARITY_CACHEDIR or singularity.cacheDir Nextflow options enables you to store and re-use the images from a central location for future pipeline runs.
   • If you are using conda, it is highly recommended to use the NXF_CONDA_CACHEDIR or conda.cacheDir settings to store the environments in a central location for future pipeline runs.

4. Start running your own analysis!

   • Typical command for CUT&Run/CUT&Tag analysis:

     nextflow run nf-core/cutandrun \
         -profile <docker/singularity/podman/conda/institute> \
         --input samplesheet.csv \
         --genome GRCh38

See usage docs for all of the available options when running the pipeline.

Documentation

The nf-core/cutandrun pipeline comes with documentation about the pipeline usage, parameters and output.

Credits

nf-core/cutandrun was originally written by Chris Cheshire (@chris-cheshire) and Charlotte West (@charlotte-west) from Luscombe Lab at The Francis Crick Institute, London, UK.

The pipeline structure and parts of the downstream analysis were adapted from the original CUT&Tag analysis protocol from the Henikoff Lab.

We thank Harshil Patel (@drpatelh) and everyone in the Luscombe Lab (@luslab) 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.

For further information or help, don't hesitate to get in touch on the Slack #cutandrun channel (you can join with this invite).

Citations

If you use nf-core/cutandrun for your analysis, please cite it using the following doi: 10.5281/zenodo.5653535

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.