Cell Ranger's pipelines analyze sequencing data produced from Chromium Single Cell Gene Expression. It also processes data generated by using Feature Barcode technology. The
count pipeline cannot be used to analyze Fixed RNA Profiling (FRP) data.
The analysis involves the following steps:
cellranger mkfastqon the Illumina BCL output folder to generate FASTQ files.
cellranger counton each GEM well that was demultiplexed by
cellranger mkfastq. If you created a Feature Barcode library alongside the Gene Expression library, you will pass them both to
cellranger countat this point. See Feature Barcode Analysis for details.
- Optionally, run
cellranger aggrto aggregate multiple GEM wells from a single experiment that were analyzed by
- Optionally run
cellranger reanalyzeto re-run the secondary analysis on a library or aggregated set of libraries (i.e., PCA, t-SNE, and clustering) and be able to fine-tune parameters. For the following example, assume that the Illumina BCL output is in a folder named
First, follow the instructions on running cellranger mkfastq to generate FASTQ files. For example, if the flow cell ID was
cellranger mkfastq will output FASTQ files in
To generate single cell feature counts for a single library, run
cellranger count with the following arguments. For a complete listing of the arguments accepted, see the Command Line Argument Reference below, or run
cellranger count --help. Cell Ranger must not be used for Single Cell Multiome Analysis. For Single Cell Multiome ATAC + Gene Expression libraries, use Cell Ranger ARC.
After determining these input arguments and customizing the code, run
cd /home/jdoe/runs cellranger count --id=sample345 \ --transcriptome=/opt/refdata-gex-GRCh38-2020-A \ --fastqs=/home/jdoe/runs/HAWT7ADXX/outs/fastq_path \ --sample=mysample \ --localcores=8 \ --localmem=64
Following a series of checks to validate input arguments,
cellranger count pipeline stages will begin to run:
Martian Runtime - v4.0.8 Running preflight checks (please wait)... Checking sample info... Checking FASTQ folder... Checking reference... Checking optional arguments... ...
By default, Cell Ranger will use all of the cores available on your system to execute pipeline stages. You can specify a different number of cores to use with the
--localcores option; for example,
--localcores=16 will limit Cell Ranger to using up to sixteen cores at once. Similarly,
--localmem will restrict the amount of memory (in GB) used by Cell Ranger.
The pipeline will create a new folder named with the sample ID you specified (e.g.
/home/jdoe/runs/sample345) for its output. If this folder already exists, Cell Ranger will assume it is an existing pipestance and attempt to resume running it.
cellranger count run should conclude with a message similar to this:
Outputs: - Run summary HTML: /opt/sample345/outs/web_summary.html - Run summary CSV: /opt/sample345/outs/metrics_summary.csv - BAM: /opt/sample345/outs/possorted_genome_bam.bam - BAM index: /opt/sample345/outs/possorted_genome_bam.bam.bai - Filtered feature-barcode matrices MEX: /opt/sample345/outs/filtered_feature_bc_matrix - Filtered feature-barcode matrices HDF5: /opt/sample345/outs/filtered_feature_bc_matrix.h5 - Unfiltered feature-barcode matrices MEX: /opt/sample345/outs/raw_feature_bc_matrix - Unfiltered feature-barcode matrices HDF5: /opt/sample345/outs/raw_feature_bc_matrix.h5 - Secondary analysis output CSV: /opt/sample345/outs/analysis - Per-molecule read information: /opt/sample345/outs/molecule_info.h5 - CRISPR-specific analysis: null - Loupe Browser file: /opt/sample345/outs/cloupe.cloupe - Feature Reference: null - Target Panel File: null Waiting 6 seconds for UI to do final refresh. Pipestance completed successfully! yyyy-mm-dd hh:mm:ss Shutting down. Saving pipestance info to "tiny/tiny.mri.tgz"
The output of the pipeline will be contained in a folder named with the sample ID you specified (e.g.
cellranger count has successfully completed, you can browse the resulting web summary HTML file in any supported web browser and open the
.cloupe file in Loupe Browser. Refer to the Understanding Outputs 3' Gene Expression Outputs page for descriptions about all output files.
A list of all Cell Ranger
count arguments are provided on the Cell Ranger manual page.