scRNA-utils

modules and utility scripts for processing scRNA data

Installation

Installation requires the use of Nextflow, a workflow description language (WDL) that enables reproducible parallelization of common bioinformatics tasks. Nextflow provides an executable that requires both Groovy and Java/JDK. Installation of the portable executable is as follows:

wget -qO- https://get.nextflow.io | bash

or

curl -s https://get.nextflow.io | bash

More specific installation instructions for Nextflow can be found here.

Pipeline installation & Updating

git clone https://github.com/GaitiLab/scRNA-utils.git
git checkout main
git pull

Modules

Modules represent individual processes for dedicated single cell tasks, such as executing cellranger to running scrublet doublet detection on a series of matrices. They are designed to be run individually, or as part of a larger workflow/pipeline.

Modules can be run using the following generic command:

nextflow run scRNA-utils/modules/{module_selection}/ where the module_selection is the name of the specific module to be run. Currently the available modules can be found in the modules directory:

• cellranger: runs cellranger count on either a directory (recursive or not) of FASTQ files, or a sample sheet with samples and their file outputs specified. See below for more information.
• epiAneuFinder: NOTE: experimental: currently not maintained. Runs an experimental scATAC-seq CNV caller on count matrices using the epiAneuFinder R package.
• fastqc_multiqc: Given a directory (recursive or not) of FASTQ files, run FastQC and multiQC (optional) on the files.
• kb-python: NOTE: experimental: currently not maintained. Runs kb-python (kallisto bustools) on a set of FASTQ files.
• scrublet: NOTE: experimental: currently not maintained. Runs scrublet for doublet detection on a count matrix output of either split-pipe or cellranger count.
• split-pipe: Process ParseBio FASTQ files into count matrices and alignment files using split-pipe --mode all and/or split-pipe --mode comb. See below for more information.

Running individual scRNA processing pipelines from modules

Within the modules directory are two basic pipelines for processing scRNA-seq data from raw FASTQ files:

• ParseBio data (to be processed using split-pipe)
• 10X Genomics data (to be processed using cellranger)

Below are the links to the specific user documentation for each type of scRNA-seq data.

Running the split pipe pipeline for ParseBio data

parseBio split-pipe analysis pipeline
split-pipe instructions using Nextflow

Running the cellranger count pipeline for 10x Genomics scRNA data

cellranger count pipeline for 10X scRNA
cellranger instructions using Nextflow

Workflows

Workflows represent more complex and linked series of processes. Currently there is one workflow in development for toggling between both ParseBio and 10X scRNA data. The workflow can be found in workflows and enables the following behaviour:

• Specifying the type of input scRNA data with --method as either split-pipe or cellranger.
• For either mode, the pipeline will generate count matrices from FASTQ files, run FastQC (and MultiQC optionally), and run scrublet on the filtered output count matrices.

Running combined/toggled scRNA processing pipelines from workflows

Currently under development.