Cell Ranger6.0, printed on 11/05/2024
Cell Ranger 6.0 introduces support for analyzing 3' Cell Multiplexing data with the cellranger multi subcommand.
The cellranger multi subcommand is required to analyze 3' Cell Multiplexing data. Otherwise, users can continue to use cellranger count.
3' GEX | 3' FB | CellPlex | Use multi? |
---|---|---|---|
Yes | Yes | Yes | Required |
Yes | Yes | No | Optional. Prefer count |
Yes | No | Yes | Required |
Yes | No | No | Optional. Prefer count |
No | Yes | No | Optional. Prefer count |
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.
Running cellranger multi requires a config CSV, described below, invoking the following arguments:
Argument | Description |
---|---|
--id | A unique run ID string: e.g. sample345 that is also the output folder name. Cannot be more than 64 characters. |
--csv | Path to config CSV file with input libraries and analysis parameters. |
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
Multi Config CSV | |
---|---|
Section: [gene-expression] | |
Field | Description |
reference | Path of folder containing 10x-compatible reference. Required for gene expression and Feature Barcode libraries. |
min-assignment-confidence | Introduced in Cell Ranger 6.0.2. The minimum estimated likelihood to call a sample as tagged with a Cell Multiplexing Oligo instead of "Unassigned". Users may wish to tolerate a higher rate of mis-assignment in order to obtain more singlets to include in their analysis, or a lower rate of mis-assignment at the cost of obtaining fewer singlets. By default, this value is 0.9. Contact [email protected] for further advice. |
cmo-set | Optional. CMO set CSV file, declaring CMO constructs and associated barcodes. |
target-panel | Optional. Path to a target panel CSV file or name of a 10x Genomics fixed gene panel (pathway, pan-cancer, immunology, neuroscience). |
no-target-umi-filter | Optional. Disable targeted UMI filtering stage. Default: false. |
r1-length | Optional. Hard trim the input Read 1 of gene expression libraries to this length before analysis. Default: do not trim Read 1. |
r2-length | Optional. Hard trim the input Read 2 of gene expression libraries to this length before analysis. Default: do not trim Read 2. |
chemistry | Optional. Assay configuration. NOTE: by default, the assay configuration is detected automatically, which is the recommended mode. Users usually will not need to specify a chemistry. Options are: 'auto' for autodetection, 'threeprime' for Single Cell 3', 'fiveprime' for Single Cell 5', 'SC3Pv1' or 'SC3Pv2' or 'SC3Pv3' for Single Cell 3' v1/v2/v3, 'SC5P-PE' or 'SC5P-R2' for Single Cell 5', paired-end/R2-only, 'SC-FB' for Single Cell Antibody-only 3' v2 or 5'. Default: auto. |
expect-cells | Optional. Expected number of recovered cells. Default: 3000. |
force-cells | Optional. Force pipeline to use this number of cells, bypassing cell detection. Default: detect cells using EmptyDrops. |
include-introns | Optional. Include intronic reads in count. Default: false |
no-secondary | Optional. Disable secondary analysis, e.g. clustering. Default: false. |
no-bam | Optional. Do not generate a bam file. Default: false. |
Section: [feature] | |
Field | Description |
reference | Feature reference CSV file, declaring Feature Barcode constructs and associated barcodes. Required for Feature Barcode libraries, otherwise optional. |
r1-length | Optional. Hard trim the input Read 1 of Feature Barcode libraries to this length before analysis. Default: do not trim Read 1. |
r2-length | Optional. Hard trim the input Read 2 of Feature Barcode libraries to this length before analysis. Default: do not trim Read 2. |
Section: [libraries] (see also Specifying Input FASTQ Files for cellranger multi) | |
Column | Description |
fastq_id | Required. The Illumina sample name to analyze. This will be as specified in the sample sheet supplied to mkfastq or bcl2fastq. |
fastqs | Required. The folder containing the FASTQ files to be analyzed. Generally, this will be the fastq_path folder generated by cellranger mkfastq. |
lanes | Optional. The lanes associated with this sample, separated by |. Defaults to using all lanes. |
feature_types | Required. The underlying feature type of the library, which must be one of ‘Gene Expression’ (3' and 5'), ‘VDJ’ (5' only), ‘VDJ-T’ (5' only), ‘VDJ-B’ (5' only), ‘Antibody Capture’ (3' and 5'), ‘CRISPR Guide Capture’ (3' only), or ‘Multiplexing Capture’ (3' only). |
subsample_rate | Optional. The rate at which reads from the provided FASTQ files are sampled. Must be strictly greater than 0 and less than or equal to 1. |
Section: [samples] | |
Column | Description |
sample_id | A name to identify a multiplexed sample. Must be alphanumeric with hyphens and/or underscores, and less than 64 characters. Required for cell multiplexing libraries. |
cmo_ids | The cell multiplexing oligo IDs used to multiplex this sample, separated by |. Required for cell multiplexing libraries. |
description | Optional. A description for the sample. |
For help on how to configure the [libraries] section to target a particular set of FASTQs, consult Specifying Input FASTQ Files for cellranger multi.
|
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.
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
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.
[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
[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
[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
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.
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