nextflow-scRNAseq

Nextflow for single-cell RNAseq

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Note: Currently please run using Conda Environment

1. Introduction

Overview:

Note: This pipeline is designed to be run post running CellRanger Count (cellrangers.nf). 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:

• DoubletFinder: Single-Cell Remover of Doublets.
• Add meta, will add the metadata from CellRanger Count and user provided samplesheet to .h5 file.
• Create QC Report of all Samples provided.
• Cell Clustering and Cell Type Annotation.
• Perform Trajectory Analysis.

2. Installation

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-7.2.0.tar.gz "https://cf.10xgenomics.com/releases/cell-exp/cellranger-7.2.0.tar.gz?Expires=1697174417&Key-Pair-Id=APKAI7S6A5RYOXBWRPDA&Signature=U496cFRNxHuNHHpEKwFQgWNq8dUjTU~Vw3bLC35tlX9rLXeYNb1sCwMkP~GYXbsMFpt8MmbIYaFkpzGZoc1UuFNcH6-wL4wOpq-2EX6xkpo3wA3DSUoOf1vQzIvGXtRnLQCCKrQcei10~jO~WCS7xrmigrfP8sFTl0r7~AKzsNiMmPaiQflW~IyvocUm-r4lGeEim0twODUypxSneiT7ddhcDmrskb5v~NbNv9NTODMbfzgjAwVknPbVPgxPNhX9i3q-uf66IbNx7jQMVGRxkWjW2iG-6OglKO4M61RgNNL2UOEKdxUb18k5l7S0-J7gAOImUtp0xrZt1IiuX0HKTA__"

To download dependencies that were developed internally by BWH Bioinformatics and Genomics Hub Note: Currently it is much easier to have conda handle the packages.

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3. Nextflow installation

To install nextflow:

git clone https://github.com/PAIN-initiative/nextflow-scRNAseq.git
cd nextflow-scRNAseq
cd nextflow
tar -xvzf nextflow-22.10.6.tar.gz
cd nextflow-22.10.6/
sudo apt install openjdk-17-jre # if on linux
./nextflow

Nextflow will be successfully installed

4. Conda

To create conda environment with dependencies installed:

cd nextflow-scRNAseq
cd env/ 
conda env create -f environment.yml
conda activate scrna_nextflow_pipeline
Rscript install_R_packages.R
pip install cellbender
cd ..
R # to activate R
install.packages('ggpubr')
# Leiden Algorithm Requirements
pip install leidenalg
pip install numpy
pip install pandas
nextflow/nextflow-22.10.6/nextflow run scrna_seq.nf

5. Containerization

To deal with software dependencies and version controling a dockerfile has been created.
To download docker image run

docker pull acicalo4/snscrnaseq:latest

To mount data from local host to docker container run, example:

docker run -t -i -v path/to/data/you/want/mounted:/container/dir acicalo4/snscrnaseq /bin/bash

6. Example Usage: To Run After Pulling Docker Image

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: \

sample_id
P1708_SP093_105

nextflow run scrna_seq.nf -with-docker acicalo4/snscrnaseq