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nextflow-scRNAseq

Nextflow for single-cell RNAseq

1. Introduction

Overview:

Note: This pipeline is designed to be run post running CellRanger Count. If the data meets basic QC from the CellRanger Websummary this pipeline will QC further and as well as run Seurat Preprocessing and Clustering.

This pipeline manages a scRNA-Seq workflow starting from raw fastq files and converting them to standard file formats for use by downstream tools. The steps involved are:

  • Add meta, will add the metadata from CellRanger Count and user provided samplesheet to .h5 file.
  • Create QC Report of all Samples provided.
  • Uses DoubletFinder to remove doublets. https://github.com/chris-mcginnis-ucsf/DoubletFinder
  • Cell Clustering and Cell Type Annotation.

Dependencies

This repository uses CellRanger Counts to generate the CellRangers outs directory that is used downstream: You can download it here: CellRanger: https://support.10xgenomics.com/single-cell-gene-expression/software/downloads/latest

You can Download CellRanger Software with this command: 
wget -O cellranger-7.1.0.tar.gz "https://cf.10xgenomics.com/releases/cell-exp/cellranger-7.1.0.tar.gz?Expires=1677814041&Policy=eyJTdGF0ZW1lbnQiOlt7IlJlc291cmNlIjoiaHR0cHM6Ly9jZi4xMHhnZW5vbWljcy5jb20vcmVsZWFzZXMvY2VsbC1leHAvY2VsbHJhbmdlci03LjEuMC50YXIuZ3oiLCJDb25kaXRpb24iOnsiRGF0ZUxlc3NUaGFuIjp7IkFXUzpFcG9jaFRpbWUiOjE2Nzc4MTQwNDF9fX1dfQ__&Signature=CeQnToHDIRIiiKInKBCYNLXM3TnZOI346o1XXSjTiPCaAO-B4r1kVheGJF3ZkWWZG1ea1DJN9P4kQ9BHzrP--PHPYhatI5gIB8pxD74WoNAxa4iZxiQAYUKRq7I4z58L2jVwgTbddeOWfSSi2atH2zUVVOOAepnmGkH554a-zdBw2wT4CX1SABsFJ9uODCKkYn5wjg~KxOAK2ULT6FAWcf6IJWLM4OKm9Lz~ill576WFYIfl3OMng~tp-MPC-i641I9mb3qB4O1rsYSvI-IrvULbJ~aCUqNqW9pkdBZxfx5RHzv-SNpSau7WYLN613UMyKshMm07W-GFtwZ~XYYU5w__&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA"

This repository requires that pandoc and libhdf5-devel libraries are installed as dependencies of the H5MANIPULATOR functions:

sudo apt-get install pandoc libhdf5-dev

CRAN packages can be installed in R using:

install.packages(c('BiocManager', 'devtools','FactoMineR', 'ggforce','systemfonts', 'assertthat', 'cowplot', 'data.table', 'dplyr', 'ids', 'ggplot2', 'jsonlite', 'Matrix', 'optparse', 'GetoptLong', 'purrr','R.utils','rmarkdown','stringr','gt','plotly','tidyr','Seurat','future','future.apply','rio','purrr','scCustomize','egg','DT','SoupX','reticulate','glmpca','remotes','viridis','qs','gridExtra','plyr','circlize','naniar','ggpubr','XML','RCurl'),repos='http://cran.us.r-project.org'); BiocManager::install(c('rhdf5', 'SingleCellExperiment', 'SummarizedExperiment','ComplexHeatmap','EnhancedVolcano'))"
# Dot plot is depedent on GitHub Report (https://github.com/Simon-Leonard/FlexDotPlot)
devtools::install_github("Simon-Leonard/FlexDotPlot")
remotes::install_github('chris-mcginnis-ucsf/DoubletFinder')
remotes::install_github('satijalab/seurat-wrappers')

Some Packages are Dependent on BiocManager

if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install("scry")
BiocManager::install("ComplexHeatmap")
BiocManager::install("rhdf5")

To download dependencies that were developed internally by BWH Bioinformatics and Genomics Hub

Sys.setenv(GITHUB_PAT = "[your_PAT_here]")
devtools::install_github("bwh-bioinformatics-hub/H5MANIPULATOR")
devtools::install_github("bwh-bioinformatics-hub/qcreporter")

git clone https://github.com/bwh-bioinformatics-hub/rna_seq_pipeline_bwh.git

Github pages of dependencies developed internally:
scRNA_seq Pipeline: https://github.com/bwh-bioinformatics-hub/rna_seq_pipeline_bwh
H5MANIPULATOR: https://github.com/bwh-bioinformatics-hub/H5MANIPULATOR
qcreporter: https://github.com/bwh-bioinformatics-hub/qcreporter \

To create conda environment with dependencies install

conda env create -f environment.yml

Setup:

Nextflow will parse a .csv file for the sample_ids and the path to the directory the fastq files are in for your project. Please provide at the minimum a sample_id column to the .csv file.
If working with a .xls/.xlsx file please create a .csv file called samples.csv with a column labeled == 'sample_id'
example:
https://github.com/bwh-bioinformatics-hub/nextflow-scRNAseq/blob/main/samplesheet/samplesheet.csv

sample_id
P1708_SP093_105