10x Genomics
Chromium Single Cell Gene Expression

Cell Ranger7.1, printed on 11/05/2024

Fixed RNA Profiling with cellranger multi

Cell Ranger v7.0 and later supports analyzing Fixed RNA Profiling (FRP) data with the cellranger multi pipeline. Cell Ranger v7.1 supports analysis of single cell FFPE (scFFPE) datasets.

Table of Contents

• Run cellranger mkfastq
• Run cellranger multi
• Command to download a customizable multi config CSV
• Example multi config CSVs
• Probe set reference CSV
• Aggr combinations

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:

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:

• The [gene-expression] section has two columns that specify parameters relevant to analysis of gene expression data, such as reference genome and cell-calling parameters that apply to the whole library in the case of singleplex Fixed RNA Profiling.
• The [feature] section has two columns that specify parameters relevant to analysis of Feature Barcode libraries; required when analyzing singleplex Fixed RNA Profiling data with Cell Surface Protein ("Antibody Capture" library).
• The [libraries] section has three required columns that specify where the input FASTQ files may be found.
• The [samples] section has two required columns that specify sample information for Fixed RNA Gene Expression with multiple samples or a single sample with multiple Probe Barcodes; it is not valid for singleplex Fixed RNA Profiling analysis.

Example formats for different product configurations are below. A template for a multi config CSV can be downloaded here or via the command line. Example multi config CSVs can be downloaded from 7.0 public datasets here.

• After determining these input arguments, run cellranger multi (replace code in red with relevant file path and ID name):

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 - v4.0.8
 
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.

• 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 outputs 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.

Command to download a customizable multi config CSV

Cell Ranger v7.1 enables users to download a multi config CSV template by running:

cellranger multi-template --output=/path/to/FILE.csv

Remember to replace code in red with the path to the directory in which you wish to output the template. Omitting the file path downloads the file into your working directory. After downloading, please remember to customize the template based on your assay and experimental design.

To print a list and description of all configurable parameters available in cellranger multi, run

cellranger multi-template --parameters

Specifying both --parameters and --output will output a parameter documentation file. Run cellranger multi-template --help or cellranger multi-template -h for more information about available flags.

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.

• Singleplex FRP, one sample with one Probe Barcode
• Singleplex FRP with Antibody Capture, one sample with one Probe Barcode
• Multiplex FRP, multiple samples with multiple Probe Barcodes per sample
• Multiplex FRP, one sample with multiple Probe Barcodes
• Multiplex FRP, multiple samples with one Probe Barcode per sample

Experimental Design	Multi 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

The v1.0.1 probe set reference CSV files for human and mouse can be found in the probe_sets directory of the Cell Ranger package (v7.1):

cellranger-7.1.0/probe_sets/
└── Chromium_Human_Transcriptome_Probe_Set_v1.0.1_GRCh38-2020-A.csv
└── Chromium_Mouse_Transcriptome_Probe_Set_v1.0.1_mm10-2020-A.csv

The v1.0.1 and v1.0 probe set reference CSV files and additional support files for human and mouse:

• Can be downloaded from the Cell Ranger Downloads page
• Are described in detail in the Probe Sets Overview

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.