nf-core/cageseq

CAGE-sequencing analysis pipeline with trimming, alignment and counting of CAGE tags.

CAGE-seq pipeline.

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 artefact trimming (cutadapt), rRNA removal (SortMeRNA, alignment to a reference genome (STAR or bowtie1) 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 comes with docker containers making installation trivial and results highly reproducible.

Quick Start

1. Install nextflow

2. Install any of Docker, Singularity or Podman 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,<docker/singularity/podman/conda/institute>

   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.

4. Start running your own analysis!

nextflow run nf-core/cageseq -profile <docker/singularity/podman/conda/institute> --input '*_R1.fastq.gz' --aligner <'star'/'bowtie1'> --genome GRCh38

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

Pipeline Summary

By default, the pipeline currently performs the following:

1. Input read QC (FastQC)
2. Adapter + EcoP15 + 5'G trimming (cutadapt)
3. (optional) rRNA filtering (SortMeRNA),
4. Trimmed and filtered read QC (FastQC)
5. Read alignment to a reference genome (STAR or bowtie1)
6. CAGE tag counting and clustering (paraclu)
7. CAGE tag clustering QC (RSeQC)
8. Present QC and visualisation for raw read, alignment and clustering results (MultiQC)

Documentation

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

Credits

nf-core/cageseq was originally written by Kevin Menden (@KevinMenden) and Tristan Kast (@TrisKast) and updated by Matthias Hörtenhuber (@mashehu).

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

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. ReadCube: Full Access Link

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):R25. 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):e0177459. doi: 10.1371/journal.pone.0177459. eCollection 2017. PubMed PMID: 28494014; PubMed Central PMCID: PMC5426675.

cageseq's Issues

Missing branch: dev and TEMPLATE

Hi,
I'm looking at all pipelines, and I noticed that this one is missing some branches.
All pipelines should have a dev branch and a TEMPLATE branch.

cageseq error

Hi

I am getting input tuple error. Could you please help me with this?

WARN: Access to undefined parameter `config_profile_contact` -- Initialise it to a default value eg. `params.config_profile_contact = some_value`
WARN: Access to undefined parameter `config_profile_url` -- Initialise it to a default value eg. `params.config_profile_url = some_value`
Run Name          : big_payne
Input             : sample_1/*R1.fastq.gz
5' artifacts      : software/cageseq/assests/artifacts_5end.fasta
3' artifacts      : software/cageseq/assests/artifacts_3end.fasta
trim_ecop         : true
trim_linker       : true
trim_5g           : true
trim_artifacts    : true
eco_site          : CAGCAG
linker_seq        : TCGTATGCCGTCTTC
Remove rRNA       : true
Aligner           : STAR
STAR Index        : hg38.analysisSet.chroms
Min. cluster      : 30
Cluster Threshold : 0.2
bigwig output     : Yes
Save Reference    : false
Max Resources     : 160 GB memory, 20 cpus, 5d 23h time per job
Output dir        : test_results/
Launch dir        : software
Working dir       : software/work
Script dir        : software/cageseq
User              : KG
Config Profile    : chimera
Config Files      : software/cageseq/nextflow.config
----------------------------------------------------
No signature of method: java.lang.Boolean.getFileSystem() is applicable for argument types: () values: []


WARN: Input tuple does not match input set cardinality declared by process `fastqc` -- offending value: sample_1/sample.R1.fastq.gz
WARN: Input tuple does not match input set cardinality declared by process `trim_adapters` -- offending value: sample_1/sample.R1.fastq.gz
[-        ] process > get_software_versions -
[-        ] process > convert_gtf           -
-[nf-core/cageseq] Pipeline completed with errors-

error: make_ctss.sh: line 18: Read-only file system Read-only file system

Hi,

I don't know if this technically qualifies as a bug but I am having issue using the pipeline on a hpcc. I don't have sudo privileges and am using singularity. I receive the error below when running with the test file (nextflow run nf-core/cageseq -r 1.0.2 -dsl1 -profile test,singularity).

make_ctss.sh: line 18: /tmp/make_ctss.sh.17128.txt: Read-only file system

My attempts at fixes that haven't worked

I have tried inserting a binding line into the Dockerfile at line 11 after the ENV PATH line. This seemed the most logical to me. I tried "exec --bind /scratch:/tmp" and "export SINGULARITY_BINDPATH=/scratch:/tmp" at this position. When this failed, I tried inserting the lines into line 12 of main.nf. I honestly did not expect this to work but figured I would try. I also tried inserting in the singularity block of nextflow.config at line 111.

Into causing error line 130 of main.nf

Operator into has been deprecated -- it's not available in DSL2 syntax

-- Check script '/rhome/aboyd003/.nextflow/assets/nf-core/cageseq/main.nf' at line: 130

Add alignment warnings for bowtie

Currently warnings for failed or low-percentage alignments are only implemented for the star alignment process.

Would be nice if we can implement this for bowtie as well.

Where are the versions numbers stored?

The link in the text below redirects to an empty page?

"First, go to the nf-core/cageseq releases page and find the latest version number - numeric only (eg. 1.3.1). Then specify this when running the pipeline with -r (one hyphen) - eg. -r 1.3.1."

release first version

Features:

• fastqc
• trimming
• trimming fastqc
• mapping
• mapping qc
• clustering cage tags
• clustering qc

Documentation:

• output.md
• usage.md
• [ ]