1. X. Shirley Liu Lab
  2. CRISPR
  3. MAGeCK-VISPR

Overview

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Quality Control, Modeling and Visualization of CRISPR/Cas9 Screens with MAGeCK-VISPR

MAGeCK-VISPR is a comprehensive quality control, analysis and visualization workflow for CRISPR/Cas9 screens. For recent changes, see the change log. The workflow combines

  • the MAGeCK algorithm to identify essential genes from CRISPR/Cas9 screens considering multiple conditions
  • with VISPR to interactively explore results and quality control in a web-based frontend.

Please visit the MAGeCK and VISPR pages for details about these tools. In particular, visit VISPR if you only want to test the interactive visualization.

Join our MAGeCK-VISPR google group if you have any questions or comments.

Tutorials

We provide a set of YouTube video tutorials to help you go through the usage of MAGeCK-VISPR (thanks to Anya Zhang). The tutorial includes videos for installing MAGeCK-VISPR, exploring quality control and comparison results, and running the whole pipeline:

Prerequisites

The input of the MAGeCK-VISPR workflow are raw FASTQ files. If you already have MAGeCK results or only want to test the interactive visualization, you can directly use VISPR and don't need to install the whole workflow. Further, it is recommended to execute this workflow on a linux server or cluster rather than your laptop or workstation, since it is computationally expensive.

Installation

To install MAGeCK-VISPR you have to use the Python 3 variant of the Miniconda Python distribution (http://conda.pydata.org/miniconda.html). MAGeCK-VISPR cannot be installed on the Python 2 variant of Miniconda. When installing Miniconda, make sure that you answer yes to this question:

Do you wish the installer to prepend the Miniconda3 install location to PATH ...? [yes|no]

Also, make sure that you do not have set the PYTHONPATH environment variable, because it will interfere with the Miniconda setup. Afterwards you have to setup Bioconda as described here (step 2). Then, you can create an isolated software environment for mageck-vispr by executing

conda create -n mageck-vispr mageck-vispr

in a terminal. The environment can be activated via

source activate mageck-vispr

To update mageck-vispr, run

conda update mageck-vispr

from within the environment.

If you are using an old version of MacOS X and the conda command is not available after installation of Miniconda, you have to change your shell to bash. To do this permanently, issue

chsh -s /bin/bash

Usage

All steps below have to be executed in a terminal.

Testing MAGeCK-VISPR

We provide test data for exploring the interactive visualization interface of VISPR at the VISPR homepage.

The MAGeCK-VISPR workflow is harder to test, because input data (raw FASTQ files) is usually big. However, we provide a subsampled test dataset based on Yusa et al. (Nat. Biotechn. 2014). While this test data won't yield reasonable results, it can be used to see how the workflow is configured and executed. There are two test datasets available:

  • configuring the workflow from scratch (Step 2, see Usage): download
  • running a configured workflow (Step 4, see Usage): download

Please choose from which step you want to test the MAGeCK-VISPR, download and extract the corresponding dataset and start with the corresponding step from the Usage section below.

For usage of MAGeCK-VISPR on real data, please see all steps below.

Step 0: Activate the mageck-vispr environment

If you have installed mageck-vispr as shown above, make sure to activate the corresponding conda environment before conducting any mageck-vispr related command via

source activate mageck-vispr

Conda environments allow you to use mutliple versions of the same software without conflicts. E.g., mageck-vispr uses Python 3.5, which might be not the default Python version in your system.

Step 1: Choosing a workflow directory

To analyze a particular set of raw FASTQ files representing a CRISPR/Cas9 screening experiment, MAGeCK-VISPR first requires you to select a directory where the workflow shall be executed and results will be stored. Please choose a meaningful name and ensure that the directory is empty.

Step 2: Initializing a new workflow

We assume that you have chosen a workflow directory (here path/to/my/workflow), and that you have a set of FASTQ files (path/to/sample1.fastq path/to/sample2.fastq ...) you want to analyze. You can initialize the workflow with

mageck-vispr init path/to/my/workflow --reads path/to/sample1.fastq path/to/sample2.fastq ...

which installs a README, a config file config.yaml and a Snakemake workflow definition (a Snakefile) to the given directory. For further information about the mageck-vispr command issue

mageck-vispr --help

for further information.

Step 3: Configure the workflow

If you provided the FASTQ files to mageck-vispr in above command, the config file already contains a mapping between putative sample names and the file paths. Now change into your workflow directory via

cd path/to/my/workflow

and open the config.yaml file. Edit the config file to your needs. Especially, define experiments for use with MAGeCK. Here, you can choose between providing treatment and control samples or a design matrix. See the MAGeCK homepage for details.

Step 4: Execute the workflow

Once configured, the workflow can be executed with Snakemake. First, it is advisable to invoke a dry-run of the workflow with

snakemake -n

which will display all jobs that will be executed. If there are errors in your config file, you will be notified by Snakemake. The actual execution of the workflow can be started with

snakemake --cores 8

here using 8 CPU cores in parallel. For more information on how to use Snakemake, refer to the Snakemake Documentation.

Step 5: Visualize results with VISPR

Once workflow execution has finished, you can visualize the generated results and quality controls by issueing

vispr server results/*.vispr.yaml

in your workflow directory. For more information and VISPR options, please visit the VISPR homepage.

Authors

License

Licensed under the MIT license (http://opensource.org/licenses/MIT). This project may not be copied, modified, or distributed except according to those terms.