Introduction

The recent advent of Next Generation Sequencing (NGS) techniques, has enabled transcriptome-scale analysis of the RNA epistructurome. Despite the establishment of several methods for querying RNA secondary structures (CIRS-seq, SHAPE-seq, Structure-seq, DMS-seq, PARS, SHAPE-MaP, DMS-MaPseq), and RNA post-transcriptional modifications (Ψ, m¹A, m⁶A, m⁵C, hm⁵C, 2'-OMe) on a transcriptome-wide scale, no tool has been developed to date to enable the rapid analysis and interpretation of this data.

RNA Framework is a modular toolkit developed to deal with RNA structure probing and post-transcriptional modifications mapping high-throughput data.
Its main features are:

• Automatic reference transcriptome creation
• Automatic reads preprocessing (adapter clipping and trimming) and mapping
• Scoring and data normalization
• Accurate RNA folding prediction by incorporating structural probing data

For updates, please visit: http://www.rnaframework.com
For support requests, please post your questions to: https://github.com/dincarnato/RNAFramework/issues

Author

Danny Incarnato (dincarnato[at]rnaframework.com)
University of Groningen

Reference

Incarnato et al., (2018) RNA Framework: an all-in-one toolkit for the analysis of RNA structures and post-transcriptional modifications (PMID: 29893890).

Incarnato et al., (2015) RNA structure framework: automated transcriptome-wide reconstruction of RNA secondary structures from high-throughput structure probing data (PMID: 26487736).

License

This program is free software, and can be redistribute and/or modified under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or any later version.

Please see http://www.gnu.org/licenses/ for more information.

Prerequisites

• Linux/Mac system
• Bowtie v1.2.3 (http://bowtie-bio.sourceforge.net/index.shtml), or
  Bowtie v2.3.5 or greater (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)
• SAMTools v1.15 or greater (http://www.htslib.org/)
• BEDTools v2.31.0 or greater (https://github.com/arq5x/bedtools2/)
• Cutadapt v2.1 or greater (http://cutadapt.readthedocs.io/en/stable/index.html)
• ViennaRNA Package v2.7.0 or greater (http://www.tbi.univie.ac.at/RNA/), with Perl bindings
• RNAstructure v6.5 or greater (http://rna.urmc.rochester.edu/RNAstructure.html) [optional]
• Perl v5.12 (or greater), with ithreads support

Optionally, for plot generation, the following are also required:

• R (https://www.r-project.org/) and the following packages:
  1. ggplot2 (https://cran.r-project.org/web/packages/ggplot2/index.html)
  2. patchwork (https://cran.r-project.org/web/packages/patchwork/index.html)
  3. RColorBrewer (https://cran.r-project.org/web/packages/RColorBrewer/index.html)

The rf-covalign module additionally requires:

• Infernal v1.1.4 or greater (http://eddylab.org/infernal/)
• R-scape v2.0.0.q or greater (http://eddylab.org/R-scape/)

Installation

Clone the RNA Framework git repository:

git clone https://github.com/dincarnato/RNAFramework

This will create the RNAFramework/ folder.
To add RNA Framework executables to your PATH, simply type:

export PATH=$PATH:/path/to/RNAFramework

Using RNA Framework via nf-core

The nf-core/rnastructurome pipeline, developed and maintained by the RNACentral team, is a reproducible, scalable workflow for analysing RNA chemical probing datasets.

It builds on RNA Framework and it follows current best practices for the analysis of RNA chemical probing data, providing a streamlined pipeline, from processing of raw sequencing data to generation of RNA reactivity profiles and RNA secondary structure predictions.

The pipeline is available at nf-core.