ChimericFragments is a tool for mapping and analyzing sequencing data from RNA-Seq experiments that produce chimeric reads. It is split into an analysis part and a Dash-based interactive visualization. ChimericFragments is highly configurable to enable analyses of different experimental workflows in different organisms and the visualization aims to provide an additional layer of accessibility to the data generated by those analyses. A running instance of the visualization of a V. cholerae RIL-seq experiment can be found here.
What ChimericFragments can do:
- Compute tabular data for interactions, multi-chimeras and single transcripts.
- Compute significance based on complementarity between ligated fragments.
- Visualize the network of interactions and host the visualization as a web app.
- Allow for searching and filtering of the network by various criteria.
- Provide interactive plots for individual and aggregated ligation events.
Necessary inputs to the analysis pipeline:
- Sequencing reads (single end or paired end, FASTA or FASTQ, gzipped or not)
- A genome (FASTA)
- A corresponding annotation for CDS (and optionally UTRs) and non-coding RNAs (GFF3)
ChimericFragments is written in the Julia programming language and requires Julia (1.10.4) to be installed on your system. You can download Julia here (old releases). It is highly recommended to install Julia as described here. ChimericFragments will not work with a Julia installation from your OS repository or a package manager like conda.
ChimericFragments depends on fastp (0.23.3+) for reads trimming and on bwa-mem2 (2.2.1+) and samtools (1.14.0+) for sequence alignments and .bam file processing. If you run ChimericFragments on Linux, the binaries are provided and do not have to be installed separately. For other operating systems (not tested), you can find the tools on github (bwa-mem2, samtools, fastp).
You can find the latest release of ChimericFragments here. To install, download the compressed source code and unzip the archive. It is tested on Linux and the default parameters of ChimericFragments are set for running on Linux and no paths to any of the binary dependencies have to be provided. For other operating systems, the paths to the binaries have to be set in the configuration file. ChimericFragments depends on a number of Julia packages that have to be installed and precompiled. This is done automatically on the first run of ChimericFragments and can take up to a few minutes.
To update ChimericFragments, download the latest version and use the new files. In general, the analysis and visualization are only compatible within one version and analysis results should be recomputed after an update.
The workflow can be split into the following steps:
- create a project folder
- copy default_config.jl to the folder and edit as necessary
- run analyze.jl on the config file to analyze your experiment
- run visualize.jl on the config file to visualize the results
IT IS IMPORTANT TO KEEP THE STRUCTURE OF config.jl INTACT. PLEASE ONLY EDIT TEXT BETWEEN "", NUMBERS OR EXCHANGE true AND false.
All relevant analysis parameters and the paths to the binaries can be set in the config.jl file. For ChimericFragments to run properly, you have to set the FILE PARAMETERS and the ANNOTATION PARAMETERS. You can find detailed descriptions of every parameter in the config file. A working example project can be found in the example_project folder.
The scripts can be executed by passing them to the julia executable. In a terminal, run
julia path/to/analyze.jl path/to/project_folder/config.jl
To rerun the analysis (e.g. with a different set of parameters), remove the results folder from the project folder and rerun the above command.
In the specified data folder, alignments (.bam, .bam.bai) and alignment stats (.bam.log) are saved. In the results folder, several subfolder are generated and all relevant results are saved in tabular format in the tables folder.
After a successful run of the analysis, the visualization can be started by running
julia path/to/visualize.jl path/to/project_folder/config.jl
The visualization can now be accessed in your browser. Under default settings, on your own machine use the address http://localhost:8050 or remotely access it by replacing localhost with the IP of the host running the visualization. To shutdown the visualization, use Ctrl-C in the terminal.
To run the analysis on the supplied example toy data, open a terminal in this folder and run
julia analyze.jl example_project/config.jl
The example analysis will take a minute and the resulting tables and data files will be located in the folder example_project/results. After the successful analysis part, you can start the visualization:
julia visualyze.jl example_project/config.jl
This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.
This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with this program. If not, see https://www.gnu.org/licenses/.
Author: Malte Siemers, Friedrich Schiller University Jena
If you use ChimericFragments or its products for your work, please cite:
Malte Siemers, Anne Lippegaus, Kai Papenfort, ChimericFragments: computation, analysis and visualization of global RNA networks, NAR Genomics and Bioinformatics, Volume 6, Issue 2, June 2024, lqae035, https://doi.org/10.1093/nargab/lqae035