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Cell Ranger


Loupe

10x Genomics
Chromium Single Cell Gene Expression

Fixed RNA Profiling with cellranger multi

Cell Ranger 7.0 supports analyzing Fixed RNA Profiling (FRP) data with the cellranger multi pipeline.

Table of Contents

Run cellranger mkfastq

First, follow the instructions on running cellranger mkfastq to generate FASTQ files. For example, if the flow cell ID 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:

ArgumentDescription
--idA unique run ID string: e.g. sample345 that is also the output folder name. Cannot be more than 64 characters.
--csvPath to config CSV file with input libraries and analysis parameters.

The multi config CSV contains both the library definitions and experimental design variables. The required sections differ slightly for analysis with single-sample ("singleplex") vs. multiplexed configurations. It is composed of up to four sections for FRP data:

Example formats for different product configurations are below. A template for a multi config CSV can be downloaded here and example multi config CSVs can be downloaded from 7.0 public datasets here.

Multi Config CSV
Section: [gene-expression]
FieldDescription
reference Required. Path of folder containing 10x Genomics-compatible genome reference.
probe-set Required. Path to the probe set reference CSV file. This file is included with the Cell Ranger 7.0 package and on the Download Links page.
filter-probes Optional. Include all non-deprecated probes listed in the probe set reference CSV file. Probes that are predicted to have off-target activity to homologous genes are excluded from analysis by default. Setting filter-probes to false will result in UMI counts from all non-deprecated probes, including those with predicted off-target activity, to be used in the analysis. Probes whose ID is prefixed with DEPRECATED are always excluded from the analysis. Default: true
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 (recommended mode). Users will typically not need to specify a chemistry. Options are: auto for auto-detection, or to override: SFRP for singleplex FRP and MFRP for multiplex FRP. Default: auto
expect-cells Optional. Override the pipeline’s auto-estimate. See cell calling algorithm overview for details on how this parameter is used. If used, enter the expected number of recovered cells. Specifying library-level expect-cells in the [gene-expression] section is only valid for the singleplex FRP configuration; note this flag name has a dash (-).
force-cells Optional. Force pipeline to use this number of cells, bypassing cell detection. Specifying library-level force-cells in the [gene-expression] section is only valid for the singleplex Fixed RNA Profiling configuration; note this flag name has a dash (-). Default: detect cells using Cell Ranger's cell calling algorithm
no-secondary Optional. Disable secondary analysis, e.g. clustering. Default: false
no-bam Optional. Set this flag to true to skip BAM file generation. If unsure, we recommend setting this option to true for Fixed RNA Profiling analysis, which will reduce the total computation time for the pipestance and the size of the output directory. Default: false
check-library-compatibility Optional.This option allows users to disable the check that evaluates 10x Barcode overlap between libraries when multiple libraries are specified (e.g., Gene Expression + Antibody Capture). Setting this option to false will disable the check across all library combinations. We recommend running this check (default), however if the pipeline errors out, users can bypass the check to generate outputs for troubleshooting. Default: true
Section: [feature]
FieldDescription
reference Path to the Feature reference CSV file, declaring Antibody Capture 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)
ColumnDescription
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. Path to the folder containing the FASTQ files to be analyzed. Generally, this will be the fastq_path folder generated by cellranger mkfastq.
feature_types Required. The underlying feature type of the library, which must be one of Gene Expression or Antibody Capture.
lanes Optional. The lanes associated with this sample, separated with a pipe (e.g., 1|2). Defaults to using all lanes.
physical_library_id Optional. Library type. NOTE: by default, the library type is detected automatically based on specified feature_types (recommended mode). Users typically do not need to include the physical_library_id column in the CSV file.
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]
ColumnDescription
sample_id Required. A name to identify a multiplexed sample or a single sample with multiple Probe Barcodes. Must be alphanumeric with hyphens and/or underscores, and less than 64 characters.
probe_barcode_ids Required. The Fixed RNA Probe Barcode IDs used for this sample. If multiple Probe Barcodes were used for a sample, separate IDs with a pipe (e.g., BC001|BC002).
description Optional. A description for the sample.
expect_cells Optional. Override the pipeline’s auto-estimate. See Gene Expression algorithm overview for details. If used, enter the expected number of recovered cells. Specifying sample-level expect_cells in the [samples] section is only valid for the multiplex Fixed RNA Profiling configuration; note this column name has an underscore (_).
force_cells Optional. Force pipeline to use this number of cells, bypassing cell detection. Specifying sample-level force_cells in the [samples] section is only valid for the multiplex Fixed RNA Profiling configuration; note this column name has an underscore (_). Default: detect cells using EmptyDrops
cd /home/jdoe/runs
cellranger multi --id=sample345 --csv=/home/jdoe/sample345.csv
Martian Runtime - v4.0.8
 
Running preflight checks (please wait)...
...

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 run ID you specified using the --id argument (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.

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"

Example multi config CSVs

Here are a few example multi config CSVs for some common Fixed RNA Profiling (FRP) assay configurations, along with simplified diagrams for the corresponding experimental set up. Make sure to replace /path/to with the actual full path to your data, and edit any text in red according to the experiment's sample/library/file names.

Important: In the examples below, we set no-bam to “true” so Cell Ranger will not generate a BAM file. This setting is recommended for Fixed RNA Profiling libraries and will reduce both the total computation time for the pipestance and the size of the output directory.

Experimental DesignMulti config CSV
Singleplex FRP, 1 Probe Barcode


See example dataset
Note: this library configuration does not use the [samples] section in the multi config CSV.
[gene-expression]
reference,/path/to/transcriptome
probe-set,/path/to/probe/set
no-bam,true #do not generate BAM file
[libraries] fastq_id,fastqs,feature_types frp_gex,/path/to/fastqs,Gene Expression
Singleplex FRP with Antibody Capture, 1 Probe Barcode


See example dataset
Antibody Capture is compatible in this configuration. There is one sample, one Probe Barcode, and two libraries (Gene Expression and Antibody Capture). Note: this library configuration does not use the [samples]section in the multi config CSV.
[gene-expression]
reference,/path/to/transcriptome
probe-set,/path/to/probe/set
no-bam,true #do not generate BAM file
[libraries] fastq_id,fastqs,feature_types frp_gex,/path/to/fastqs,Gene Expression frp_ab,/path/to/fastqs,Antibody Capture
[feature] reference,/path/to/feature_reference.csv
Multiplex FRP, multiple Probe Barcodes/sample
A. Multiple biological samples

[gene-expression]
reference,/path/to/transcriptome
probe-set,/path/to/probe/set
no-bam,true #do not generate BAM file
[libraries] fastq_id,fastqs,feature_types frp_gex,/path/to/fastqs,Gene Expression
[samples] sample_id,probe_barcode_ids,description sample1,BC001|BC002,Control sample2,BC003|BC004,Treated
B. Single biological sample

In this case, the config CSV must include a [samples] section to specify the Probe Barcodes since two Probe Barcodes were used for a single sample in this experiment. Note that this configuration is not compatible with Antibody Capture.
[gene-expression]
reference,/path/to/transcriptome
probe-set,/path/to/probe/set
no-bam,true #do not generate BAM file
[libraries] fastq_id,fastqs,feature_types frp_gex,/path/to/fastqs,Gene Expression
[samples] sample_id,probe_barcode_ids,description sample1,BC001|BC002,Control
Multiplex FRP, 1 Probe Barcode/sample


See example dataset
Note that this configuration is not compatible with Antibody Capture.
[gene-expression]
reference,/path/to/transcriptome
probe-set,/path/to/probe/set
no-bam,true #do not generate BAM file
[libraries] fastq_id,fastqs,feature_types frp_gex,/path/to/fastqs,Gene Expression
[samples] sample_id,probe_barcode_ids,description sample1,BC001,Control sample2,BC003,Treated

Probe set reference CSV

Probe set reference CSV file for human can be found in the probe_sets directory in the Cell Ranger package:

cellranger-7.0.0/probe_sets/
└── Chromium_Human_Transcriptome_Probe_Set_v1.0_GRCh38-2020-A.csv

The probe set reference CSV file and additional support files for human:

Aggr combinations

The following Gene Expression (GEX) assay chemistry combinations may be aggregated with cellranger aggr, and are either supported (validated by 10x Genomics) or possible (will run, but not the validated intended usage). Targeted Gene Expression is not compatible for aggregation with Fixed RNA Gene Expression (FRP). Some assay chemistry combinations are supported for aggregation as long as they have the same feature reference CSV file (+) and/or probe set reference CSV file (^); please see this FAQ page for details about aggregating these assay combinations.

Assay chemistry Singleplex FRP Singleplex FRP, Antibody Multiplex FRP
Singleplex FRP Supported^ - -
Singleplex FRP, Antibody Supported+^ Supported+^ -
Multiplex FRP Supported^ Supported+^ Supported^
3' or 5' GEX library Possible Possible+^ Possible