Subgenomic RNAs Profile ANalysis (SPAN): Profiling of Noncanonical Subgenomic RNAs in SARS-CoV-2 Variants.

Description:

• A SgRNA Profile ANalysis (SPAN) is proposed for profiling various subgenomic RNAs (sgRNAs) and accompany with SPANviewer for data visualization. This pipeline is currently under submission in the manuscript titled "Profiling of Noncanonical Subgenomic RNAs in SARS-CoV-2 Variants".
• This pipeline first maps the reads to the SARS-CoV-2 reference genome and generates junction coordinates from splice alignments. The junction coordinates are then processed using a Python script to identify subgenomic RNAs and finally visualized using R Shiny. All scripts can be run independently.

Requirements:

1. Linux or MacOS
2. R (version 4.1)
3. python (version 3.8.10)

Installation for SPAN:

Environments setup:

conda env create -f ./SPAN.yml -n SPAN
conda activate SPAN

Data visualization in our R shiny app (Plot_Profiling.R):

• Before using this app, please install R packages first.

Rscript install.R

Three pages we provided are as follows.

1. Venn Diagram page
2. Junction Site Sashimi Plot page
3. Boxplot page

Usage:

1. Activate conda environment:

   conda activate SPAN
   

2. Please prepare fastq files in ~/SPAN/{variant_name} folder (e.g., use SRAToolkit fasterq-dump function to retrieve SRA reads)
3. Generate sample.txt:

   python ./scripts/generate_sample_list.py
   

4. Execute this SPAN pipeline with snakemake:

   snakemake -c1
   

5. Data visualization:

   Rscript ./R/SPANviewer.R
   

Authors @ Chang Gung University

• Yu-Nong Gong
• Chia-Te Wang