Introduction

nf-core/nottocode is a bioinformatics designed to annotate long non-coding RNAs (lncRNAs) from transcriptome assemblies. It works by filtering, comparing, and characterizing transcripts from GTF files, which can be generated by tools such as stringtie via nf-core/rnaseq.

The pipeline performs the following key steps:

• Filtering assembled transcripts by expression (TPM >= 1)

• Merging assemblies across samples

• Classifying transcripts based on coding potential

• Annotating protein domains in predicted open reading frames (ORFs)

• Generating reports with MultiQC

It supports a modular design and can be easily extended or integrated into custom analyses.

Modules and Tools

This pipeline uses a combination of nf-core and custom modules:

• samplesheet_check

• gtffiltertpm

• stringtie/merge

• gffcompare

• compare_transcriptomes

• gffread

• mstrg

• cpc2

• plek

• transdecoder/longorfs

• hmmer/hmmpress, hmmer/hmmsearch

• multiqc

Workflow Overview

```mermaid graph TD A[Samplesheet input] --> B[TPM filter] B --> C[StringTie merge] C --> D[GffCompare] D --> E[Transcript comparison] E --> F[GffRead] F --> G[MSTRG formatting] G --> H[CPC2 and PLEK - coding potential] H --> I[TransDecoder - long ORFs] I --> J[HMMER - Pfam scan] J --> K[MultiQC and reports]

```

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,gtf CONTROL_REP1,AEG588A1.gtf

Each row represents a gtf file.

Now, you can run the pipeline using:

bash nextflow run nf-core/nottocode \ --input /path/to/samplesheet.csv \ --reference_gtf /path/to/genome.gff \ --reference_genome /path/to/genome.fna \ --outdir results \ --pfam_db /path/to/Pfam-A.hmm \

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/nottocode was developed by @gleisonm and @rafaellaferraz.

We thank all contributors to the nf-core community for support and feedback.

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

Citations

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.