10x Genomics
Chromium Single Cell Gene Expression

Cell Ranger6.0, printed on 12/21/2024

Cell Multiplexing with cellranger multi

Cell Ranger 6.0 introduces support for analyzing 3' Cell Multiplexing data with the cellranger multi subcommand.

Table of Contents

1. When to use the multi pipeline
2. Run cellranger mkfastq
3. Run cellranger multi
4. Output files
5. Example multi config CSVs
6. CMO Reference

When to use the multi pipeline

The cellranger multi subcommand is required to analyze 3' Cell Multiplexing data. Otherwise, users can continue to use cellranger count.

Run cellranger mkfastq

First, follow the instructions on running cellranger mkfastq to generate FASTQ files. For example, if the flowcell serial number was HAWT7ADXX, then cellranger mkfastq will output FASTQ files in HAWT7ADXX/outs/fastq_path. If you are already starting with FASTQ files, you can skip this step and proceed directly to run cellranger multi.

Run cellranger multi

Running cellranger multi requires a config CSV, described below, invoking the following arguments:

The multi config CSV contains both the library definitions and experimental design variables. It is composed of up to three sections for 3' data: [gene-expression], [feature], and [libraries]. The [gene-expression] and [feature] sections have at most two columns, and are responsible for configuring their respective portions of the experiment. The [libraries] section specifies where input FASTQ files may be found. A template for a multi config CSV can be downloaded here, and example multi config CSVs can be downloaded from 6.0 public datasets here

After determining these input arguments, run cellranger:

$ cd /home/jdoe/runs
$ cellranger multi --id=sample345 --csv=/home/jdoe/sample345.csv

Following a series of checks to validate input arguments, cellranger multi pipeline stages will begin to run:

Martian Runtime - v4.0.4
 
Running preflight checks (please wait)...
...

By default, cellranger 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 cellranger to using up to sixteen cores at once. Similarly, --localmem will restrict the amount of memory (in GB) used by cellranger.

The pipeline will create a new folder named with the run ID you specified using the --id argument (e.g. /home/jdoe/runs/sample345) for its output. If this folder already exists, cellranger will assume it is an existing pipestance and attempt to resume running it.

Output Files

A successful cellranger multi run should conclude with a message similar to this:

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 run ID you specified (e.g. sample345). The subfolder named outs will contain the main pipeline outputs

Example multi config CSVs

Here are a few example multi config CSVs examples for some common product configurations. Make sure to replace /path/to with the actual PATH to the data.

3' Gene Expression with Cell Multiplexing

[gene-expression]
reference,/path/to/transcriptome

[libraries]
fastq_id,fastqs,feature_types
gex1,/path/to/fastqs,Gene Expression
mux1,/path/to/fastqs,Multiplexing Capture

[samples]
sample_id,cmo_ids
sample1,CMO301|CMO302
sample2,CMO303|CMO304

3' Gene Expression with Feature Barcoding and Cell Multiplexing

[gene-expression]
reference,/path/to/transcriptome

[libraries]
fastq_id,fastqs,feature_types
gex1,/path/to/fastqs,Gene Expression
abc1,/path/to/fastqs,Antibody Capture
mux1,/path/to/fastqs,Multiplexing Capture

[samples]
sample_id,cmo_ids
sample1,CMO301|CMO302
sample2,CMO303|CMO304

3' Gene Expression with CRISPR and Cell Multiplexing

[gene-expression]
reference,/path/to/transcriptome

[libraries]
fastq_id,fastqs,feature_types
gex1,/path/to/fastqs,Gene Expression
csp1,/path/to/fastqs,CRISPR Guide Capture
mux1,/path/to/fastqs,Multiplexing Capture

[samples]
sample_id,cmo_ids
sample1,CMO301|CMO302
sample2,CMO303|CMO304

CMO Reference

The cmo-set option to the [gene-expression] table of the multi config CSV allows you provide a reference for custom multiplexing oligos. The design of this reference is identical to the Feature Barcode Reference used to describe feature barcodes, with one difference: the feature_type is required to be Multiplexing Capture instead of those feature types supported in the feature barcode reference.

Default CMO Reference

id,name,read,pattern,sequence,feature_type
CMO301,CMO301,R2,5P(BC),ATGAGGAATTCCTGC,Multiplexing Capture
CMO302,CMO302,R2,5P(BC),CATGCCAATAGAGCG,Multiplexing Capture
CMO303,CMO303,R2,5P(BC),CCGTCGTCCAAGCAT,Multiplexing Capture
CMO304,CMO304,R2,5P(BC),AACGTTAATCACTCA,Multiplexing Capture
CMO305,CMO305,R2,5P(BC),CGCGATATGGTCGGA,Multiplexing Capture
CMO306,CMO306,R2,5P(BC),AAGATGAGGTCTGTG,Multiplexing Capture
CMO307,CMO307,R2,5P(BC),AAGCTCGTTGGAAGA,Multiplexing Capture
CMO308,CMO308,R2,5P(BC),CGGATTCCACATCAT,Multiplexing Capture
CMO309,CMO309,R2,5P(BC),GTTGATCTATAACAG,Multiplexing Capture
CMO310,CMO310,R2,5P(BC),GCAGGAGGTATCAAT,Multiplexing Capture
CMO311,CMO311,R2,5P(BC),GAATCGTGATTCTTC,Multiplexing Capture
CMO312,CMO312,R2,5P(BC),ACATGGTCAACGCTG,Multiplexing Capture