nf-core/cageseq

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Introduction

nf-core/cageseq is a bioinformatics analysis pipeline used for CAGE-seq sequencing data.

The pipeline takes raw demultiplexed fastq-files as input and includes steps for linker and artifact (including 5’G) trimming (cutadapt), rRNA removal (SortMeRNA), alignment to a reference genome (STAR or bowtie) and CAGE tag counting and clustering (paraclu). Additionally, several quality control steps (FastQC, RSeQC, MultiQC) are included to allow for easy verification of the results after a run.

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!

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.

Pipeline summary

1. Input read QC (FastQC)
2. Adapter + EcoP15 + 5’G + artifact fragments trimming (cutadapt)
3. (optional) rRNA filtering (SortMeRNA),
4. Trimmed and filtered read QC (FastQC)
5. Read alignment to a reference genome (STAR or bowtie)
6. CAGE tag counting and clustering (paraclu)
7. CAGE tag cluster QC (RSeQC)
8. Present QC and visualisation for raw read, alignment and clustering results (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/cageseq -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!

   nextflow run nf-core/cageseq -profile <docker/singularity/podman/shifter/charliecloud/conda/institute> --input samplesheet.csv --genome GRCh37

Documentation

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

Credits

nf-core/cageseq was originally written by Kevin Menden, Tristan Kast, Matthias Hörtenhuber.

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 #cageseq channel (you can join with this invite).

Citations

If you use nf-core/cageseq for your analysis, please cite it using the following doi: 10.5281/zenodo.4095105

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.

In addition, references of tools and data used in this pipeline are as follows:

Nextflow

Di Tommaso P, Chatzou M, Floden EW, Barja PP, Palumbo E, Notredame C. Nextflow enables reproducible computational workflows. Nat Biotechnol. 2017 Apr 11;35(4):316-319. doi: 10.1038/nbt.3820. PubMed PMID: 28398311.

Pipeline tools

• BEDTools

  Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010 Mar 15;26(6):841-2. doi: 10.1093/bioinformatics/btq033. Epub 2010 Jan 28. PubMed PMID: 20110278; PubMed Central PMCID: PMC2832824.

• bowtie

  Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10(3)

  . doi: 10.1186/gb-2009-10-3-r25. Epub 2009 Mar 4. PMID: 19261174; PMCID: PMC2690996.

• cutadapt

  Martin, M., 2011. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. journal, 17(1), pp.10-12.

• FastQC

• MultiQC

  Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct 1;32(19):3047-8. doi: 10.1093/bioinformatics/btw354. Epub 2016 Jun 16. PubMed PMID: 27312411; PubMed Central PMCID: PMC5039924.

• paraclu

  Frith MC, Valen E, Krogh A, Hayashizaki Y, Carninci P, Sandelin A. A code for transcription initiation in mammalian genomes. Genome Res. 2008 Jan;18(1):1-12. doi: 10.1101/gr.6831208. Epub 2007 Nov 21. PMID: 18032727; PMCID: PMC2134772.

• RSeQC

  Wang L, Wang S, Li W. RSeQC: quality control of RNA-seq experiments Bioinformatics. 2012 Aug 15;28(16):2184-5. doi: 10.1093/bioinformatics/bts356. Epub 2012 Jun 27. PubMed PMID: 22743226.

• SAMtools

  Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009 Aug 15;25(16):2078-9. doi: 10.1093/bioinformatics/btp352. Epub 2009 Jun 8. PubMed PMID: 19505943; PubMed Central PMCID: PMC2723002.

• SortMeRNA

  Kopylova E, Noé L, Touzet H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data Bioinformatics. 2012 Dec 15;28(24):3211-7. doi: 10.1093/bioinformatics/bts611. Epub 2012 Oct 15. PubMed PMID: 23071270.

• STAR

  Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner Bioinformatics. 2013 Jan 1;29(1):15-21. doi: 10.1093/bioinformatics/bts635. Epub 2012 Oct 25. PubMed PMID: 23104886; PubMed Central PMCID: PMC3530905.

• UCSC tools

  Kent WJ, Zweig AS, Barber G, Hinrichs AS, Karolchik D. BigWig and BigBed: enabling browsing of large distributed datasets. Bioinformatics. 2010 Sep 1;26(17):2204-7. doi: 10.1093/bioinformatics/btq351. Epub 2010 Jul 17. PubMed PMID: 20639541; PubMed Central PMCID: PMC2922891.

Software packaging/containerisation tools

• Anaconda

  Anaconda Software Distribution. Computer software. Vers. 2-2.4.0. Anaconda, Nov. 2016. Web.

• Bioconda

  Grüning B, Dale R, Sjödin A, Chapman BA, Rowe J, Tomkins-Tinch CH, Valieris R, Köster J; Bioconda Team. Bioconda: sustainable and comprehensive software distribution for the life sciences. Nat Methods. 2018 Jul;15(7):475-476. doi: 10.1038/s41592-018-0046-7. PubMed PMID: 29967506.

• BioContainers

  da Veiga Leprevost F, Grüning B, Aflitos SA, Röst HL, Uszkoreit J, Barsnes H, Vaudel M, Moreno P, Gatto L, Weber J, Bai M, Jimenez RC, Sachsenberg T, Pfeuffer J, Alvarez RV, Griss J, Nesvizhskii AI, Perez-Riverol Y. BioContainers: an open-source and community-driven framework for software standardization. Bioinformatics. 2017 Aug 15;33(16):2580-2582. doi: 10.1093/bioinformatics/btx192. PubMed PMID: 28379341; PubMed Central PMCID: PMC5870671.

• Docker

• Singularity

  Kurtzer GM, Sochat V, Bauer MW. Singularity: Scientific containers for mobility of compute. PLoS One. 2017 May 11;12(5)

  . doi: 10.1371/journal.pone.0177459. eCollection 2017. PubMed PMID: 28494014; PubMed Central PMCID: PMC5426675.