Stagger-Seq CRISPR Screen Mapping Pipeline

This a nextflow pipeline to map reads from a stagger sequencing run using the sgcount mapping tool.

Installation

Then you will need to install sgcount and fxtools.

These can be installed with the rust package manager cargo, which can be installed with the following one-liner:

You will then need to install nextflow.

Install cargo

curl --proto '=https' --tlsv1.2 -sSf https://sh.rustup.rs | sh

Install sgcount and fxtools

cargo install sgcount fxtools

Install nextflow

First make sure you have java 11 or later installed

java -version

Then download nextflow

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

This will download nextflow into your current working directory and you can validate it works with:

./nextflow run hello

You should put nextflow into your $PATH. I won't describe how to do that here but there are plenty of tutorials online on how to do so.

I am assuming that nextflow is in your $PATH for the remainder of this tutorial.

Usage

Downloading Pipeline

You can clone this git repo to get a copy for each new run.

# clone the repo
git clone \
    https://github.com/noamteyssier/stagger_seq_crispr_screen_nextflow \
    my_sequencing_run

# enter the directory
cd my_sequencing_run

Configuration

There are few things to configure, but you can make adjustments by editing the bundled file nextflow.config.

Configure CRISPR Library

You will need to specify your CRISPR library path and the gene to sgRNA (g2s) file path.

You can edit the file nextflow.config to update this.

The two variables to change are library_path and g2s_path.

Adapter

The stagger sequencing has a constant adapter region before the variable region of the library.

This adapter's position can be considered dynamically placed with a variable number of nucleotides before it.

In the data I've seen the adapter was ACCTTGTTGG. However, if you have a different adapter you can update the variable adapter in the config to reflect that.

Data

We then need to place our sequencing reads into the data/ directory bundled with this repo.

These are expected to be fastqs of the form data/<sample_name>_R1*.fastq.gz.

Execution

To run the pipeline we can use the following command:

nextflow run -resume Pipeline.nf

Outputs

All outputs of the pipeline will be available in the results/ directory that will be created.