Introduction

nf-core/marsseq is a bioinformatics single-cell preprocessing pipeline for MARS-seq v2.0 experiments. MARS-seq is a plate-based technique that can be combined with FACS in order to study rare populations of cells. On top of the pre-existing pipeline, we have developed an RNA velocity workflow that can be used to study cell dynamics using StarSolo. We do so by converting the raw FASTQ reads into 10X v2 format.

Usage

Note If you are new to Nextflow and nf-core, please refer to this page on how to set-up Nextflow. Make sure to test your setup with -profile test before running the workflow on actual data.

To run the pipeline you have create experiment metadata files:

• amp_batches.xlsx
• wells_cells.xlsx
• seq_batches.xslx

and samplesheet (samplesheet.csv). We provide test example here.

Next, you have to generate genome references to incorporate ERCC spike-ins. References are downloaded from GENCODE database.

nextflow run nf-core/marsseq \
  -profile <docker/singularity/.../institute> \
  --genome <mm10,mm9,GRCh38_v43> \
  --build_references \
  --input samplsheet.csv \
  --outdir <OUTDIR>

Now, you can run the pipeline using:

nextflow run nf-core/marsseq \
  -profile <docker/singularity/.../institute> \
  --genome <mm10,mm9,GRCh38_v43> \
  --input samplesheet.csv \
  --outdir <OUTDIR>

Warning: Please provide pipeline parameters via the CLI or Nextflow -params-file option. Custom config files including those provided by the -c Nextflow option can be used to provide any configuration except for parameters; see docs.

For more details and further functionality, please refer to the usage documentation and the parameter documentation.

Pipeline output

To see the results of an example test run with a full size dataset refer to the results tab on the nf-core website pipeline page. For more details about the output files and reports, please refer to the output documentation.

Credits

nf-core/marsseq was originally written by Martin Proks.

We thank the following people for their extensive assistance in the development of this pipeline:

• Jose Alejandro Romero Herrera (@joseale2310)
• Maxime Garcia (@maxulysse)

Keren-Shaul, H., Kenigsberg, E., Jaitin, D.A. et al. MARS-seq2.0: an experimental and analytical pipeline for indexed sorting combined with single-cell RNA sequencing. Nat Protoc 14, 1841–1862 (2019). https://doi.org/10.1038/s41596-019-0164-4

Contributions and Support

If you would like to contribute to this pipeline, please see the contributing guidelines.

For further information or help, don't hesitate to get in touch on the Slack #marsseq channel (you can join with this invite).

Citations

If you use nf-core/marsseq for your analysis, please cite it using the following doi: 10.5281/zenodo.8063539

An extensive list of references for the tools used by the pipeline can be found in the CITATIONS.md file.

You can cite the nf-core publication as follows:

The nf-core framework for community-curated bioinformatics pipelines.

Philip Ewels, Alexander Peltzer, Sven Fillinger, Harshil Patel, Johannes Alneberg, Andreas Wilm, Maxime Ulysse Garcia, Paolo Di Tommaso & Sven Nahnsen.

Nat Biotechnol. 2020 Feb 13. doi: 10.1038/s41587-020-0439-x.