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Introduction

nf-core/nascent is a bioinformatics best-practice analysis pipeline for nascent transcript (NT) and Transcriptional Start Site (TSS) assays.

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. Read QC (FastQC)
2. Adapter and quality trimming (fastp)
3. Alignment
   1. bwa
   2. bwamem2
   3. DRAGMAP
4. Sort and index alignments (SAMtools)
5. UMI-based deduplication (UMI-tools)
6. Duplicate read marking (picard MarkDuplicates)
7. Quality Control
   1. RSeQC - Various RNA-seq QC metrics
   2. Preseq - Estimation of library complexity
   3. BBMap - Analyzes the sequencing coverage
8. Coverage Graphs
   1. Create bedGraph coverage files (BEDTools
   2. Create bigWig coverage files (deeptools)
9. Transcript identification
   1. HOMER
   2. GroHMM
   3. PINTS
10. Quantification of Genes and Nascent Transcripts (featureCounts)
11. Aggregate report describing results and QC from the whole pipeline (MultiQC)

Usage

Note

If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

Now, you can run the pipeline using:

nextflow run nf-core/nascent \
   -profile <docker/singularity/.../institute> \
   --input samplesheet.csv \
   --outdir <OUTDIR>

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.

For more details and further functionality, please refer to the usage documentation and the parameter documentation.

Pipeline output

To see the results of an example test run with a full size dataset refer to the results tab on the nf-core website pipeline page. For more details about the output files and reports, please refer to the output documentation.

Credits

nf-core/nascent was originally written by Ignacio Tripodi (@ignaciot) and Margaret Gruca (@magruca).

The pipeline was re-written in Nextflow DSL2 by Edmund Miller (@Emiller88) and Sruthi Suresh (@sruthipsuresh) from The Functional Genomics Laboratory at The Univeristy of Texas at Dallas

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

• @apeltzer
• @ewels
• @drpatelh
• @pditommaso
• @FriederikeHanssen
• Tae Hoon Kim
• @easterwoods

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

Citations

If you use nf-core/nascent for your analysis, please cite it using the following doi: 10.5281/zenodo.7245273

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.