Minigene Pipeline

Single nucleotide variants (SNVs) were introduced into the mutagenesis region, spanning both introns and exons of a minigene plasmid containing the target exon. Additionally, random 12 bp barcodes, flanked by a constant 8 bp prefix and suffix, were also incorporated into the plasmid's exon. The modified minigene plasmid was then transfected into iPSCs and HEK cells and sequenced post-transcription using ONT. The pipeline first identifies barcode-SNV relationship before undergoing optional secondary splicing analyses, which includes the processing of ONT cDNA data. Statistical reports in CSVs and visualisations in PDFs of the different isoforms found as a result of different SNVs are generated as the final output files. The workflow is illustrated below.

Pre-Requisites

• Python 3.12
  • Ensure that Python 3.12 is installed on your system. Install it otherwise before proceeding.
• Singularity image of DeepVariant (v1.6.1 (CPU version))

  BIN_VERSION="1.6.1"
  singularity pull docker://google/deepvariant:"${BIN_VERSION}"
  

Installation of Minigene Pipeline

1. Navigate into directory where minigene pipeline is to be installed in

   wget https://github.com/AshleyLab/minigene_pipeline/releases/download/v1.0/minigene_pipeline.tar.gz
   tar -xvzf minigene_pipeline.tar.gz
   

Set Up Minigene Pipeline's Environment

2. Navigate into minigene_pipeline directory

   cd minigene_pipeline
   

3. Create and activate minigene_env

   python3.12 -m venv minigene_env      # create new python virtual env
   source minigene_env/bin/activate     # activate minigene_env
   

4. Install packages

   python3 setup_minigene_env.py 
   

Configure minigene_config.yaml file

5. Edit minigene_config.yaml file to suit your data

   vim minigene_config.yaml
   
   # press 'i' to edit and make changes; when done, press 'esc', ':wq' and hit 'enter'
   

Execution

6. For splice analysis:

   python3 run_minigene_splice.py
   

Tools

samtools: samtools
chopper: chopper
minimap2: minimap2
deepvariant: deepvariant
GMAP: GMAP