nf-core pipeline · facs-sorting, mars-seq, single-cell
nf-core/marsseq
Preprocessing pipeline for MARS-seq v2 single-cell RNA sequencing data with RNA velocity estimation via StarSolo.
What nf-core/marsseq is
nf-core/marsseq is a bioinformatics pipeline designed to process single-cell RNA sequencing (scRNA-seq) data generated using the MARS-seq v2.0 protocol. MARS-seq is a plate-based single-cell assay frequently coupled with fluorescence-activated cell sorting (FACS) to capture and characterise rare cell populations. The pipeline was originally written by Martin Proks, with contributions from Jose Alejandro Romero Herrera and Maxime Garcia.
Beyond primary preprocessing, the pipeline incorporates an RNA velocity workflow to analyse transcriptional dynamics across single cells. It achieves this by reformatting MARS-seq sequence reads into 10X Genomics v2 structure, allowing standard processing through StarSolo to quantify spliced and unspliced transcripts alongside cell-level expression.
What the pipeline does
The pipeline ingests raw FASTQ sequencing files alongside tabular experiment metadata. If requested, it downloads reference data from the GENCODE database and builds reference genomes incorporating external RNA control consortium (ERCC) spike-in controls.
During execution, raw MARS-seq FASTQ reads are converted into the 10X Genomics v2 format. The reformatted reads are then aligned and quantified using StarSolo, which computes expression matrices and facilitates downstream RNA velocity analysis by separating spliced and unspliced reads.
Key facts
- Pipeline engine
- Nextflow
- Target protocol
- MARS-seq v2.0
- Alignment and velocity tool
- StarSolo
- Input formats
- FASTQ, XLSX, CSV
- Annotation source
- GENCODE
- Original developer
- Martin Proks
- Latest release
- 1.0.3
What you can use it for
- MARS-seq v2.0 data processing
- Demultiplex and quantify single-cell gene expression libraries derived from MARS-seq v2 plate-based assays.
- Transcriptional dynamics and RNA velocity
- Estimate unspliced and spliced transcript abundance in single cells using StarSolo to evaluate developmental trajectories and cell state changes.
- Analysis of FACS-sorted rare cell populations
- Process single-cell transcriptomes isolated via index-sorting flow cytometry to investigate low-abundance cell types.
Run it here
The research agent runs nf-core/marsseq with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
Strengths and limits
Good at
- Extends MARS-seq v2 processing to support RNA velocity calculations.
- Converts reads into 10X Genomics v2 layout to leverage StarSolo for fast alignment and quantification.
- Automates the construction of reference indices containing ERCC spike-in controls from GENCODE sources.
Watch out for
- Requires three specific Excel metadata files (amp_batches, wells_cells, seq_batches) in addition to the standard samplesheet.
- Restricted to data produced according to the MARS-seq v2.0 sequencing scheme.
Questions
Can I run nf-core/marsseq without my own cluster?
The research agent runs nf-core/marsseq with Nextflow on its own machine — up to 32 cores, 256 GiB of memory and 24 hours per run, which covers the test profile and real single-sample runs. It builds the samplesheet, checks it against the pipeline's schema and reads the results back. When a run is too big for one machine, it gives you the exact command for your own cluster instead.
What licence is nf-core/marsseq under?
MIT, like every nf-core pipeline.
What metadata files are required to run the workflow?
Runs require a samplesheet in CSV format along with three Excel spreadsheets: amp_batches.xlsx, wells_cells.xlsx, and seq_batches.xlsx.
How does the pipeline enable RNA velocity analysis on MARS-seq data?
It converts raw MARS-seq reads into 10X Genomics v2 format, which allows StarSolo to process the data and quantify spliced and unspliced reads.
Can the pipeline prepare its own genome indices?
Yes. Passing the reference-building parameter instructs the workflow to download reference files from GENCODE and generate indices containing ERCC spike-ins.
How to cite it
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
Written from nf-core/marsseq's own README. Statements citing a figure that does not appear there are removed before publication. Licence and running details come from our registry.