nf-core/polyamod

Introduction

nf-core/polya+mod is a bioinformatics pipeline that enables joint prediction of poly(A) tail lengths and m6A modifications from ONT direct RNA sequencing data.

This pipeline was implemented in Nextflow (v24.10.5), a domain-specific workflow management system optimized for scalable and reproducible bioinformatics workflows. It uses Docker/Singularity containers making installation trivial and results highly reproducible.

Pipeline Summary

polya+mod automates the simultaneous prediction of poly(A) tail lengths and m6A RNA modifications at the transcript- and gene-level.

Workflow steps:

1. Basecalling - Performed using Guppy to convert raw signal data to FASTQ

2. Preprocessing - FASTQ files are converted to FASTA - f5c is used for indexing

3. Alignment - Reads are aligned to user-provided reference genome using minimap2 - Resulting BAM files are sorted and indexed using SAMtools

4. Downstream Analysis (Two Parallel Paths): - Nanopolish-polyA: Predictions poly(A) tail lengths - f5c eventalign + m6Anet: Detected m6A modifications

5. Annotation: - A provided GTF file is used to annotate results - Final outputs include: - BED file of m6A modification sites - BED file of polyadenylated transcripts

Outputs are compatible with genome browsers and can be used in exploratory analyses, such as correlating m6A presence with poly(A) tail length.

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.

First, prepare a samplesheet with your input data that looks as follows:

samplesheet.csv:

csv sample,fast5_dir,flowcell_id,sequencing_kit,reference_genome CELL_LINE_1,/path/to/fast5/directory/fast5_files,FLO-MIN106,SQK-RNA002,/path/to/reference/genome/Homo_sapiens.GRCh38.cdna.all.fa,/path/to/gtf/file/Homo_sapiens.GRCh38.113_transcripts.gtf CELL_LINE_2,/path/to/fast5/directory/fast5_files,FLO-MIN106,SQK-RNA002,/path/to/reference/genome/Homo_sapiens.GRCh38.cdna.all.fa,/path/to/gtf/file/Homo_sapiens.GRCh38.113_transcripts.gtf

Each row represents a study, containing a directory of fast5 files, flowcell ID, and sequencing kit for basecalling, as well as a reference genome and gene annotation file.

Now, you can run the pipeline using:

bash nextflow run nf-core/polyamod \ -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.

Credits

nf-core/polyamod was originally written by Sahiti Somalraju.

We thank the following people for their extensive assistance in the development of this pipeline: - David Schaeper ()

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.

This pipeline uses code and infrastructure developed and maintained by the nf-core community, reused here under the MIT license.

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.