iSCAP - An Integrated Single Cell Analysis Pipeline

Snakemake pipeline for Single Cell RNA-seq analysis. The integrated Single Cell Analysis Pipeline (iSCAP) enables combining different methods including quality control, preprocessing, normalization, batch effect correction, clustering, marker gene and subpopulation identification, cell type annotation, multi-sample differential expression analysis.

The iSCAP allows users to select and apply their preferred combination of the software tools supported in the pipeline and define sample groups for comparison analysis. iSCAP also includes gene set enrichment and network analysis, it generates HTML-based nozzle reports which allow users to browse the results obtained in each stage of the pipeline.

Prerequisites

• Software packages

  Install all the following R packages:

  cluster
  clusterProfiler
  dplyr
  igraph
  MASS
  Matrix
  org.Hs.eg.db   
  org.Mm.eg.db
  pagoda2
  RColorBrewer
  reshape2
  scales
  scater
  scClustViz
  scran
  Seurat (version 2)
  SingleR
  URD
  velocyto.R
  viridis
  

• Please install all the reference files:

  mm10:
         transcriptome = "path_to_cell_ranger_download/refdata-gex-mm10-2020-A"
  gtf = "path_to_cell_ranger_download/refdata-gex-mm10-2020-A/genes/genes.gtf"
         vgtf10x = "path_to_cell_ranger_download/RefGenomes/10X_REF/mm10_rmsk.gtf"
        
  hg19:
         transcriptome = "path_to_cell_ranger_download/refdata-cellranger-hg19-3.0.0/"
         gtf = "path_to_cell_ranger_download/refdata-cellranger-hg19-3.0.0/genes/genes.gtf"
  hg38:
         transcriptome = "path_to_cell_ranger_download/refdata-gex-GRCh38-2020-A"
         gtf = "path_to_cell_ranger_download/refdata-gex-GRCh38-2020-A/genes/genes.gtf"
         vgtf10x = "path_to_cellranger_download/RefGenomes/10X_REF/hg38_rmsk.gtf"
  

Software Installation

Please download and install all software tools acoording to the specific tool installation instruction. Please download and install the snakemake file from this software package. The snakefile will need to be altered to point to the actual package installation locations.

cellranger = "path_to_cell_ranger_download/cellranger-9.0.1/cellranger-cs/9.0.1/bin/cellranger"

velocyto = "path_to_velocyto_tools/velocyto/bin/velocyto"

Once the config file is filled in, then submit submit.sh as a job to run snakemake.

qsub submit.sh

Workflow

The following graphic gives an overview of the entire RNASeq workflow:

Running the pipeline

More detailed information about how to run the pipeline can be found on the wiki.

Contact

CCRSF_IFX@nih.gov