Installation
To use the scRNAbox pipeline, the folowing must be installed on your High-Performance Computing (HPC) system:
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
(v0.1.35) run the following command:
wget https://github.com/neurobioinfo/scrnabox/releases/download/v0.1.35/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:
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 just step 2, 2-4, run steps 2 through 4)
optional arguments:
-h (--help) = See helps regarding the pipeline options.
--method = Choose what scRNA method you want to use; use HTO and SCRNA for for hashtag nad Standard scRNA, respectively.
--nFeature_RNA_L = Lower threshold of number of unique RNA transcripts for each cell, it filters nFeature_RNA > nFeature_RNA_L.
--nFeature_RNA_U = Upper threshold of number of unique RNA transcripts for each cell, it filters --nFeature_RNA_U.
--nCount_RNA_L = Lower threshold for nCount_RNA, it filters nCount_RNA > nCount_RNA_L
--nCount_RNA_U = Upper threshold for nCount_RNA, it filters nCount_RNA < nCount_RNA_U
--mitochondria_percent_L = Lower threshold for the amount of mitochondrial transcript, it is in percent, mitochondria_percent > mitochondria_percent_L.
--mitochondria_percent_U = Upper threshold for the amount of mitochondrial transcript, it is in percent, mitochondria_percent < mitochondria_percent_U.
--log10GenesPerUMI_U = Upper threshold for the log number of genes per UMI for each cell, it is in percent,log10GenesPerUMI=log10(nFeature_RNA)/log10(nCount_RNA). mitochondria_percent < log10GenesPerUMI_U.
--log10GenesPerUMI_L = Lower threshold for the log number of genes per UMI for each cell, log10GenesPerUMI=log10(nFeature_RNA)/log10(nCount_RNA). mitochondria_percent > log10GenesPerUMI_L.
--msd = you can get the hashtag labels by running the following code
--marker = Find marker.
--sinfo = Do you need sample info?
--fta = FindTransferAnchors
--enrich = Annotation
--dgelist = creates a DGEListobject from a table of counts obtained from seurate objects.
--genotype = Run the genotype contrast.
--celltype = Run the Genotype-cell contrast.
--cont = You can directly call the contrast to the pipeline.
--seulist = You can directly call the list of seurat objects to the pipeline.
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
Users must first install R
onto their HPC system:
# install R
module load r/4.2.1
Then, users should create a designated directory on their HPC system where the required R packages will be installed:
# make common R library
mkdir R_library
cd R_library
# open R
R
# set common R library path
R_LIB_PATH="/pathway/to/R_library"
.libPaths(R_LIB_PATH)
# load packages
library(Seurat)
library(ggplot2)
library(dplyr)
library(foreach)
library(doParallel)
library(Matrix)
library(DoubletFinder)
library(cowplot)
library(clustree)
library(xlsx)
library(enrichR)
library(stringi)
library(limma)
library(tidyverse)
library(edgeR)
library(vctrs)
library(RColorBrewer)
library(fossil)
library(openxlsx)
library(stringr)
library(ggpubr)
devtools::install_github(“neurobioinfo/scrnabox/scrnaboxR”)
Upon completing the installation procedures, users can proceed with the scRNAbox pipeline using either the Standard scRNAseq Analysis Track or Cell Hashtag scRNAseq Analysis Track.