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10x Genomics
Chromium Single Cell Gene Expression

What is Cell Ranger?

Cell Ranger is a set of analysis pipelines that process Chromium single-cell RNA-seq output to align reads, generate feature-barcode matrices and perform clustering and gene expression analysis. Cell Ranger includes four pipelines relevant to single-cell gene expression experiments:

These pipelines combine Chromium-specific algorithms with the widely used RNA-seq aligner STAR. Output is delivered in standard BAM, MEX, CSV, HDF5 and HTML formats that are augmented with cellular information.

Workflows

If you are beginning with raw base call (BCL) files, the Cell Ranger workflow starts with demultiplexing the BCL files for each flowcell directory. 10x recommends using cellranger mkfastq as described in Generating FASTQs. If you are beginning with FASTQ files that have already been demultiplexed with bcl2fastq directly, or from a public source such as SRA, you can skip cellranger mkfastq and begin with cellranger count. Please see the Specifying Input FASTQs page for specific guidelines on which arguments to use for your scenario.

The exact steps of the workflow vary depending on how many samples, GEM wells, and flowcells you have. This section describes the different possible workflows.

One Sample, One GEM Well, One Flowcell

cellranger basic

In this example you have one sample that is processed through one GEM well (a set of partitioned cells from a single 10x Chromium™ Chip channel) and sequenced on one flowcell. In this case you would generate FASTQs using cellranger mkfastq, and run cellranger count as described in Single-Sample Analysis.

This example also illustrates two sequencing libraries. A single GEM well can yield multiple libraries: one Gene Expression library, and one or more Feature Barcoding libraries.

One Sample, One GEM well, Multiple Flowcells

cellranger multiple sequencing runs

In this example you have one sample that is processed through one GEM well then you generate one library which is sequenced across multiple flowcells. This may be done to increase sequencing depth, for example. In this case all of the reads can be combined in a single instance of the cellranger count pipeline. This process is described in Specifying Input Fastqs.

One Sample, Multiple GEM Wells, One Flowcell

cellranger multiple libraries

In this example you have one sample that is processed through multiple GEM wells. This is often done when conducting technical replicate experiments. The libraries from the GEM wells are then pooled onto one flowcell and sequenced. In this case you demultiplex the data from the sequencing run and then run the libraries from each GEM well through a separate instance of cellranger count. Once those are completed, you can perform a combined analysis using cellranger aggr, as described in Multi-Library Aggregation. (See figure above.)

Multiple Samples, Multiple GEM Well, One Flowcell

cellranger multiple samples

In this example you have multiple samples that are processed through multiple GEM wells which generate multiple libraries and are pooled onto one flowcell. In this case, after demultiplexing, you must run cellranger count separately for each GEM well. For instance, if your experiment involves two samples then you will have to run cellranger count two times. Then you can aggregate them with a single instance of cellranger aggr, as described in Multi-Library Aggregation.

One or more 5′ Gene Expression and V(D)J Enriched Libraries from the Same Biological Sample

cellranger vdj and gene expression

5′ gene expression libraries and V(D)J enriched libraries generated from the same cDNA product must be processed by cellranger count and cellranger vdj respectively. The outputs can be analyzed interactively using Loupe Cell Browser and Loupe V(D)J Browser. Please refer to Single Cell V(D)J + 5′ Gene Expression for more information.

Chemistry

Cell Ranger 3.0 is required for processing Single Cell 3' v3 libraries, or Feature Barcoding libraries. The assay support of Cell Ranger 3.0, and the previous Cell Ranger 2.2 is summarized below.

Assay TypeCell Ranger 3.0Cell Ranger 2.2
Single Cell 3' v3YesNo
Single Cell 3' v3 + Feature BarcodingYesNo
Single Cell 5'YesYes
Single Cell 5' + Feature BarcodingYesNo
Single Cell 3' v2YesYes
Single Cell 3' v2 + Feature BarcodingYesNo