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scdrake

scdrake is a scalable and reproducible pipeline for secondary analysis of droplet-based single-cell RNA-seq data. scdrake is an R package built on top of the drake package, a Make-like pipeline toolkit for R language.

The main features of the scdrake pipeline are:

  • Import of scRNA-seq data: 10x Genomics Cell Ranger output, delimited table, or SingleCellExperiment object.
  • Quality control and filtering of cells and genes, removal of empty droplets.
  • Higly variable genes detection, cell cycle scoring, normalization, clustering, and dimensionality reduction.
  • Cell type annotation.
  • Integration of multiple datasets.
  • Computation of cluster markers and differentially expressed genes between clusters (denoted as “contrasts”).
  • Rich graphical and HTML outputs based on customizable RMarkdown documents.
    • You can find links to example outputs here.
  • Thanks to drake, the pipeline is highly efficient, scalable and reproducible, and also extendable.
    • Want to change some parameter? No problem! Only parts of the pipeline which changed will rerun, while up-to-date ones will be skipped.
    • Want to reuse the intermediate results for your own analyses? No problem! The pipeline has smartly defined checkpoints which can be loaded from a drake cache.
    • Want to extend the pipeline? No problem! The pipeline definition is just an R object which can be arbitrarily extended.

For whom is scdrake purposed? It is primarily intended for tech-savvy users (bioinformaticians), who pass on the results (reports, images) to non-technical persons (biologists). At the same time, bioinformaticians can quickly react to biologists’ needs by changing the parameters of the pipeline, which then efficiently skips already finished parts. This dialogue between the biologist and the bioinformatician is indispensable during scRNA-seq data analysis. scdrake ensures that this communication is performed in an effective and reproducible manner.

The pipeline structure along with diagrams and links to outputs is described in vignette("pipeline_overview") (link).

If you use scdrake in your research, please, consider citing

Kubovciak J, Kolar M, Novotny J (2023). “Scdrake: a reproducible and scalable pipeline for scRNA-seq data analysis.” Bioinformatics Advances, 3(1). doi:10.1093/bioadv/vbad089.

Huge thanks go to the authors of the Orchestrating Single-Cell Analysis with Bioconductor book on whose methods and recommendations is scdrake largely based.

Installation instructions

A Docker image based on the official Bioconductor image (version 3.15) is available. This is the most handy and reproducible way how to use scdrake as all the dependencies are already installed and their versions are fixed. In addition, the parent Bioconductor image comes bundled with RStudio Server.

The complete guide to the usage of scdrake’s Docker image can be found in the Docker vignette. We strongly recommend to go through even if you are an experienced Docker user. Below you can find just the basic command to download the image and to run a detached container with RStudio in Docker or to run scdrake in Singularity.

You can also run the image in SingularityCE (without RStudio) - see the Singularity section in the Docker vignette above. If the image is already downloaded in the local Docker storage, you can use singularity pull docker-daemon:<image>

You can pull the Docker image with the latest stable scdrake version using

docker pull jirinovo/scdrake:1.5.2
singularity pull docker:jirinovo/scdrake:1.5.2

or list available versions in our Docker Hub repository.

For the latest development version use

docker pull jirinovo/scdrake:latest
singularity pull docker:jirinovo/scdrake:latest

Note for Mac users with M1/M2 chipsets: until version 1.5.0 (inclusive), arm64 images are available.

docker pull jirinovo/scdrake:1.5.0-bioc3.15-arm64

Running the container

For the most common cases of host machines: Linux running Docker Engine, and Windows or MacOS running Docker Desktop.

First make a shared directory that will be mounted to the container:

mkdir ~/scdrake_projects
cd ~/scdrake_projects

And run the image that will expose RStudio Server on port 8787 on your host:

docker run -d \
  -v $(pwd):/home/rstudio/scdrake_projects \
  -p 8787:8787 \
  -e USERID=$(id -u) \
  -e GROUPID=$(id -g) \
  -e PASSWORD=1234 \
  jirinovo/scdrake:1.5.2

For Singularity, also make shared directories and execute the container (“run and forget”):

mkdir -p ~/scdrake_singularity
cd ~/scdrake_singularity
mkdir -p home/${USER} scdrake_projects
singularity exec \
    -e \
    --no-home \
    --bind "home/${USER}/:/home/${USER},scdrake_projects/:/home/${USER}/scdrake_projects" \
    --pwd "/home/${USER}/scdrake_projects" \
    path/to/scdrake_image.sif \
    scdrake <args> <command>
Click for details

Install the required system packages

  • For Linux, follow the commands for your distribution here.
  • For MacOS: $ brew install libxml2 imagemagick@6 harfbuzz fribidi libgit2 geos pandoc

Install R >= 4.2

See https://cloud.r-project.org/

From now on, all commands are for R.

Install {renv}

{renv} is an R package for management of local R libraries. It is intended to be used on a per-project basis, i.e. each project should use its own library of R packages.

Initialize a new {renv} library

Switch to directory where you will analyze data and initialize a new renv library:

renv::consent(TRUE)
renv::init()

Now exit and run again R. You should see a message that renv library has been activated.

Install BiocManager 

renv::install("BiocManager")

Install Bioconductor 3.15 

BiocManager::install(version = "3.15")

Restore {scdrake} dependencies from lockfile

renv also allows to export the current installed versions of R packages (and other things) into a lockfile. Such lockfile is available for scdrake and you can use it to install all dependencies by

## -- This is a lockfile for the latest stable version of scdrake.
download.file("https://raw.githubusercontent.com/bioinfocz/scdrake/1.5.2/renv.lock")
## -- You can increase the number of CPU cores to speed up the installation.
options(Ncpus = 2)
renv::restore(lockfile = "renv.lock", repos = BiocManager::repositories())

For the lockfile for the latest development version use

download.file("https://raw.githubusercontent.com/bioinfocz/scdrake/main/renv.lock")

Install the {scdrake} package

Now we can finally install the scdrake package, but using a non-standard approach - without its dependencies (which are already installed from the lockfile).

remotes::install_github(
  "bioinfocz/scdrake@1.5.2",
  dependencies = FALSE, upgrade = FALSE,
  keep_source = TRUE, build_vignettes = TRUE,
  repos = BiocManager::repositories()
)

For the latest development version use "bioinfocz/scdrake".

Install the command line interface (CLI)

Optionally, you can install scdrake’s CLI scripts with

scdrake::install_cli()

CLI should be now accessible as a scdrake command. By default, the CLI is installed into ~/.local/bin, which is usually present in the PATH environment variable. In case it isn’t, just add to your ~/.bashrc: export PATH="${HOME}/.local/bin:${PATH}"

Every time you will be using the CLI make sure your current working directory is inside an renv project. You can read the reasons below.

Show details

You might notice that a per-project renv library and an installed CLI are “disconnected” and if you install scdrake and its CLI within multiple projects (renv libraries), then the CLI scripts in ~/.local/bin will be overwritten each time. But when you run the scdrake command inside an renv project, the renv directory is automatically detected and the renv library is activated by renv::load(), so the proper, locally installed scdrake package is then used.

Also, there is a built-in guard: the version of the CLI must match the version of the bundled CLI scripts inside the installed scdrake package. Anyway, we think changes in the CLI won’t be very frequent, so this shouldn’t be a problem most of the time.

TIP: To save time and space, you can symlink the renv/library directory to multiple scdrake projects.

Quickstart

First run the scdrake image in Docker or Singularity - see the Docker vignette

Then you can go through the Get Started vignette

Vignettes and other readings

See https://bioinfocz.github.io/scdrake for a documentation website of the latest stable version (1.5.2) where links to vignettes below become real :-)

See https://bioinfocz.github.io/scdrake/dev for a documentation website of the current development version.

We encourage all users to read basics of the drake package. While it is not necessary to know all drake internals to successfully run the scdrake pipeline, its knowledge is a plus. You can read the minimum basics in vignette("drake_basics").

Also, the prior knowledge of Bioconductor and its classes (especially the SingleCellExperiment) is considerable.

Citation

Below is the citation output from using citation("scdrake") in R. Please run this yourself to check for any updates on how to cite scdrake.

print(citation("scdrake"), bibtex = TRUE)
To cite package ‘scdrake’ in publications use:

  Jiri Novotny and Jan Kubovciak (2021). scdrake: A Pipeline For 10x Chromium Single-Cell RNA-seq Data Analysis.
  https://github.com/bioinfocz/scdrake, https://bioinfocz.github.io/scdrake.

A BibTeX entry for LaTeX users is

  @Manual{,
    title = {scdrake: A Pipeline For 10x Chromium Single-Cell RNA-seq Data Analysis},
    author = {Jiri Novotny and Jan Kubovciak},
    year = {2021},
    note = {https://github.com/bioinfocz/scdrake, https://bioinfocz.github.io/scdrake},
  }

Please note that the scdrake was only made possible thanks to many other R and bioinformatics software authors, which are cited either in the vignettes and/or the paper(s) describing this package.

Help and support

In case of any problems or suggestions, please, open a new issue. We will be happy to answer your questions, integrate new ideas, or resolve any problems 😊

You can also use GitHub Discussions, mainly for topics not related to development (bugs, feature requests etc.), but if you need e.g. a general help.

Contribution

If you want to contribute to scdrake, read the contribution guide, please. All pull requests are welcome! 🙂

Code of Conduct

Please note that the scdrake project is released with a Contributor Code of Conduct. By contributing to this project, you agree to abide by its terms.

Acknowledgements

Funding

This work was supported by ELIXIR CZ research infrastructure project (MEYS Grant No: LM2018131) including access to computing and storage facilities.

Software and methods used by {scdrake}

Many things are used by scdrake, but these are really worth mentioning:

Development tools

This package was developed using {biocthis}.