Installation

To use the scRNAbox pipeline, the folowing must be installed on your High-Performance Computing (HPC) system:

• scrnabox.slurm
• CellRanger
• R and R packages

---

scrnabox.slurm installation

scrnabox.slurm is written in bash and can be used with any Slurm system. To download the latest version of scrnabox.slurm (v0.1.52.50) run the following command:

wget https://github.com/neurobioinfo/scrnabox/releases/download/v0.1.52.5/scrnabox.slurm.zip
unzip scrnabox.slurm.zip

For a description of the options for running scrnabox.slurm run the following command:

bash /pathway/to/scrnabox.slurm/launch_scrnabox.sh -h 

If the scrnabox.slurm has been installed properly, the above command should return the folllowing:

scrnabox pipeline version 0.1.52.50
------------------- 
mandatory arguments:
                -d  (--dir)  = Working directory (where all the outputs will be printed) (give full path)
                --steps  =  Specify what steps, e.g., 2 to run step 2. 2-6, run steps 2 through 6

        optional arguments:
                -h  (--help)  = See helps regarding the pipeline arguments. 
                --method  = Select your preferred method: HTO and SCRNA for hashtag, and Standard scRNA, respectively. 
                --msd  = You can get the hashtag labels by running the following code (HTO Step 4). 
                --markergsea  = Identify marker genes for each cluster and run marker gene set enrichment analysis (GSEA) using EnrichR libraries (Step 7). 
                --knownmarkers  = Profile the individual or aggregated expression of known marker genes. 
                --referenceannotation  = Generate annotation predictions based on the annotations of a reference Seurat object (Step 7). 
                --annotate  = Add clustering annotations to Seurat object metadata (Step 7). 
                --addmeta  = Add metadata columns to the Seurat object (Step 8). 
                --rundge  = Perform differential gene expression contrasts (Step 8). 
                --seulist  = You can directly call the list of Seurat objects to the pipeline. 
                --rcheck  = You can identify which libraries are not installed.  

 ------------------- 
 For a comprehensive help, visit  https://neurobioinfo.github.io/scrnabox/site/ for documentation. 

---

CellRanger installation

For information regarding the installation of CellRanger, please visit the 10X Genomics documentation. If CellRanger is already installed on your HPC system, you may skip the CellRanger installation procedures.

---

R library preparation and R package installation

R must be installed on the HPC system. If R is not already installed, the user should contact their system administrator. If R is installed on the HPC system, users must load R (v4.2 or later) into their working environment:

# load R
module load r/4.2.1

Then, users must run the following installation code, which will create a directory where the R packages will be loaded and will install the required R packages:

# Folder for R packages 
R_PATH=~/path/to/R/library
mkdir -p $R_PATH

# Install package
Rscript ./scrnabox.slurm/soft/R/install_packages.R $R_PATH

Alternatively, users can install the packages manually. The R packages required for scRNAbox are shown here.

---

Upon completing the installation procedures, users can proceed with the scRNAbox pipeline.