MAGIC - Minigene Assembly and Generation of Isoform Combinations toolkit

A comprehensive toolkit to unravel RNA isoform structures obtained from minigene splicing assay and massive parallel sequencing, integrating both an intuitive GUI and a powerful Snakemake pipeline.

Table of Contents

• Overview
• Features
• Installation
  • GUI Component
  • Pipeline Component
• Usage
  • Using the GUI
  • Running the Snakemake Pipeline
• SOSTAR annotation
  • Output file
  • Nomenclature
• Test the toolkit
• Author
• License

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Overview

This tool is composed of two main components:

1. Graphical User Interface (GUI): A user-friendly application to generate the reference files required for the pipeline of the artificial genome representing the (multi-)exonic minigene splicing construction in fasta and gtf formats.
2. Snakemake Pipeline: A robust and scalable workflow designed for high-throughput environments.

These components can be used independently or together, depending on the user's needs.

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Features

• Easy-to-use GUI for configuration file creation
• Modular and reproducible Snakemake pipeline
• Cross-platform compatibility
• Support for containerized execution using [Docker]

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Installation

GUI Component

The GUI application for Windows is available as a standalone executable.

📦 Download the latest release here

To install:

1. Download the .exe file from the Releases section.
2. Run the executable directly (no installation required).

Pipeline Component

This pipeline is adapted from the SOSTAR pipeline. First, sequencing files were aligned using the minimap2 RNAseq aligner in either short or long read mode depending on the option used. Then, isoform combinations were assembled using the StringTie tool in a guided mode with the construction.gtf created by the GUI. They are merged using the merged mode of StringTie et expressions were computed with the expression mode of StringTie. Finally, isoforms were annotated using the SOSTAR nomenclature related to the construction used.

The Snakemake pipeline requires a Unix-like environment (Linux, macOS).

Requirements:

• Python >= 3.8
• Snakemake >= 7.32.4 or greater
• Apptainer/Singularity >= 3.5.3 or greater

The workflow automatically uses a docker image which contains the other tools required.

To set up:

git clone https://github.com/LBGC-CFB/MAGIC.git
cd ./MAGIC

To configure this workflow, modify the ./config/config.yaml file according to your needs.

• constructiondir: Path of the construction directories that contains the different constructions with their corresponding construction.fasta and construction.gtf.

• indir: Path of the directory containing either the *.fastq file for long read sequencing or the *_L001_R1_001.fastq.gz and *_L001_R2_001.fastq.gz files for short read sequencing.

• outdir: Path of the outdirectory.

• samples: names of the differents samples: name of the construction used

• threads: number of threads to use.

• option: shortread or longread, indicated by true or false.

Usage

Using the GUI

1. Launch the GUI application.

2. Define the necessary parameters.

3. Click on Generate files. The files will be generated once all the necessary parameters have been defined.

4. Transfer the file to the machine where the pipeline will be executed.

Running the Snakemake pipeline

Given that the workflow has been properly configured, it can be executed as follows:

cd ./workflow
snakemake --use-singularity --cores

You can provide your own configuration file using the --configfile option when launching the pipeline — as long as it follows the same structure and format.

⚠️ Warning: If you encounter write access errors with Apptainer/Singularity, ensure your working directories are correctly bound by exporting the proper APPTAINER_BINDPATH or SINGULARITY_BINDPATH as follow:

export APPTAINER_BINDPATH="/path/to/data/dir"

Output directory tree:

MAGIC/outdir
├── alignment
│   ├── {sample}.minimap2.bam
│   ├── {sample}.minimap2.bam.bai
│   └── ...
├── assembly
│   └── {construction}
│       ├── {sample}_assembly.gtf
│       ├── ...
│       └── {construction}_merged.gtf
│   └── ...
├── expression
│   ├── {sample}_expression.gtf
│   └── ...
├── constructions_all.gtf
└── MAGIC_SOSTAR_annotation_table_results.xlsx

• alignment folder: contains all aligned and sorted {sample} <.bam> with their corresponding index <.bai>.
• assembly folder: contains subfolders of all assembled <.gtf> {sample} with StringTie and the corresponding merge <.gtf> file for each different construction.
• expression folder: contains all assembled <.gtf> with expression metrics computed by StringTie.
• constructions_all.gtf: a global gtf file of all the different file of constructions used for the merge step.
• MAGIC_SOSTAR_annotation_table_results.xlsx: final output file containing descriptive annotation and expression metrics of each transcript in the cohort (see SOSTAR annotation section for more informations).

SOSTAR annotation

MAGIC uses the SOSTAR tool to provide annotation of all the different isoforms assembled by StringTie.

Output file

SOSTAR generates a spreadsheet file in <.xlsx> format:

transcript_id	construction	gene	annot_ref	annot_find	MIN01	MIN02	MIN03	occurence	P_MIN01	P_MIN02	P_MIN03
NM_000059_pCAS2_BRCA2_Ex3	pCAS2_BRCA2_Ex3	BRCA2	3	3	76093,1	1114,3	15320,6	3	98,76	0,6	10,53
M_pCAS2_BRCA2_Ex3.1.1	pCAS2_BRCA2_Ex3	BRCA2	3	Δ3	954	184159,5	130188,3	3	1,24	99,4	89,47

• transcript_id : transcript identifier
• construction : construction name
• gene : gene associated to the transcript
• annot_ref : reference annotation of construction
• annot_find : SOSTAR annotation of transcript (see section SOSTAR nomenclature for more details).
• sample : transcript coverage
• occurence : number of transcript occurrences in cohort
• P_sample : transcript coverage proportion between all isoform combinations

Nomenclature

Isoforms are described relative to reference transcripts (provided by user) by an annotation including only the alternative splicing events. Some conventions were established to annotate the alternative splicing events:

symbol	definition
∆	skipping of a reference exon
▼	inclusion of a reference intron
p	shift of an acceptor site
q	shift of a donnor site
(37)	number of skipped or retained nucleotides
[p23, q59]	relative positions of new splice sites
exo	exonization of an intronic sequence
int	intronization of an exonic sequence
-	continuous event
,	discontinuous event

Nomenclature example: Black boxes: exon, black lines: intron, red boxes: exon (or part of exon) skipping, green boxes: novel exon (or part of exon).

Test the toolkit

A minimal test dataset is provided in the test/ directory.

Generates genome files with MAGIC GUI

• Select your output directory.
• Use the pCAS2-51D-Ex2_9_assembledseq.fasta file as input file. This corresponds to the complete sequence of a multi-exonic minigene splicing assay of the RAD51D gene spanning exons 2 to 9.
• Complete the other parameters with thoses mentionned in parameters_for_MAGIC_GUI.yaml file.
• Click Validate and Generate Files to create the .fasta and .gtf files.

Get annotation with MAGIC pipeline

The config.yaml config file in the config directory has been completed to ensure the pipeline launches correctly with the test files.

Place the files generated by the GUI in the tests/pCAS2_RAD51D_Ex2-9 folder.

Define your own directory for output results by modifying the outdir: parameter.

Launch the pipeline using the following command line:

snakemake --use-singularity --cores

Author

Camille AUCOUTURIER @AUCAM

License

This project is under GPL-3.0 License, see LICENCE for more details.