Cell Ranger relies on a specific multi config CSV structure for proper execution. Failure to comply with the required format (e.g., column headers, delimiters) will lead to parsing errors.
Ensure that the multi config is saved in CSV format with the CSV extension.
This page provides specific details on setting up the multi config CSV for analyzing Universal 3' and 5' Gene Expression and Feature Barcode (Antibody Capture, CRISPR Guide Capture, and Antigen Capture) libraries using the cellranger multi pipeline.
For general information on setting up and running the multi pipeline, visit the Cell Ranger multi pipeline page. Go to the Cell Ranger Multi Config CSV page for a complete list of options for each section.
To generate a multi config CSV template, run cellranger multi-template and see the usage instructions here.
Examples of multi config CSVs for the most common library combinations are provided here. If your specific library combination is not listed and you need assistance, please contact 10x Genomics Support at support@10xgenomics.com.
A self-directed tutorial is available
[vdj]
reference,/path/to/vdj_reference
[libraries]
fastq_id,fastqs,feature_types
VDJ_B_fastqs_id,/path/to/vdj_B_fastqs,VDJ-B
A self-directed tutorial is available
[gene-expression]
reference,/path/to/transcriptome
create-bam,true
tenx-cloud-token-path,/path/to/10xcloud_token.json
cell-annotation-model,auto
[vdj]
reference,/path/to/vdj_reference
[libraries]
fastq_id,fastqs,feature_types
GEX_fastqs_id,/path/to/GEX_fastqs,Gene Expression
VDJ_B_fastqs_id,/path/to/vdj_B_fastqs,VDJ-B
To learn how to generate and access your 10x Cloud Analysis token, visit the cellranger annotate page.
[gene-expression]
reference,/path/to/transcriptome
create-bam,true
[vdj]
reference,/path/to/vdj_reference
[feature]
reference,/path/to/feature_ref.csv
[libraries]
fastq_id,fastqs,lanes,feature_types
GEX_fastqs_id_1,/path/to/GEX1_fastqs,1,Gene Expression
GEX_fastqs_id_2,/path/to/GEX2_fastqs,2,Gene Expression
GEX_fastqs_id_3,/path/to/GEX3_fastqs,3,Gene Expression
VDJ_B_fastqs_id_1,/path/to/vdj_B_fastqs,1,VDJ-B
VDJ_B_fastqs_id_2,/path/to/vdj_B_fastqs,2,VDJ-B
VDJ_B_fastqs_id_3,/path/to/vdj_B_fastqs,4,VDJ-B
FASTQ IDs can be extracted from the output folder names generated by bcl-convert. Specifically, the ID is the portion of the name preceding _S1, as seen in the format [Sample Name]_S1_L00[Lane Number]_[Read Type]_001.fastq.gz.
[gene-expression]
reference,/path/to/transcriptome
create-bam,true
[vdj]
reference,/path/to/vdj_reference
[feature]
reference,/path/to/feature_ref.csv
[libraries]
fastq_id,fastqs,lanes,feature_types
GEX_fastqs_id,/path/to/GEX_fastqs,1|2,Gene Expression
VDJ_B_fastqs_id,/path/to/vdj_B_fastqs,1|2,VDJ-B
VDJ_T_fastqs_id,/path/to/vdj_T_fastqs,1|2,VDJ-T
FB_fastqs_id,/path/to/FB_fastqs,1|2,Antibody Capture
CRISPR_fastqs_id,/path/to/CRISPR_fastqs,1|2,CRISPR Guide Capture
This template also applies to V(D)J + FB (without GEX) libraries. The [gene-expression] reference section is required. However, the GEX FASTQ specification under the [libraries] section must be removed for the VDJ+FB library combinations.
[gene-expression]
reference,/path/to/transcriptome
create-bam,true
tenx-cloud-token-path,/path/to/10xcloud_token.json
cell-annotation-model,auto
[libraries]
fastq_id,fastqs,feature_types
GEX_fastqs_id,/path/to/GEX_fastqs,Gene Expression
[gene-expression]
reference,/path/to/transcriptome
create-bam,true
[feature]
reference,/path/to/feature_ref.csv
[libraries]
fastq_id,fastqs,lanes,feature_types
GEX_fastqs_id,/path/to/GEX_fastqs,1|2,Gene Expression
FB_fastqs_id,/path/to/FB_fastqs,1|2,Antibody Capture
[gene-expression]
reference,/path/to/transcriptome
create-bam,true
[feature]
reference,/path/to/feature_ref.csv
[libraries]
fastq_id,fastqs,lanes,feature_types
GEX_fastqs_id,/path/to/GEX_fastqs,1|2,Gene Expression
FB_fastqs_id,/path/to/FB_fastqs,1|2,Antibody Capture
CRISPR_fastqs_id,/path/to/CRISPR_fastqs,1|2,CRISPR Guide Capture
Visit the Antigen Capture page for specific information on how to run Cell Ranger multi to analyze your Antigen Capture/BEAM libraries.
Note: BEAM is unsupported with GEM-X chemistry; therefore, Cell Ranger does not support BEAM/Antigen Capture libraries produced with this chemistry.
Cell Ranger multi v7.0.0 and later allows users to analyze T cell libraries enriched for gamma (TRG) and delta (TRD) chains. 10x Genomics does not provide reagents or primers for TRG/D chain enrichment. Since this workflow is not fully supported, the Cell Ranger pipeline has not been extensively tested for TRG/D libraries, and the algorithm's performance cannot be guaranteed.
To analyze TRG/D libraries, set feature_types to VDJ-T-GD in the [libraries] section of the multi config CSV. Auto-detection does not work for TRG/D chains. If set to auto-detection, TRG/D libraries are treated as VDJ-T libraries enriched for alpha-beta chains, and the gamma-delta chains are filtered out. The pipeline runs to completion, but zero barcodes are assigned to cells.
Refer to the example multi config CSV for additional configuration guidance. Outputs from a successful gamma-delta run are located in the vdj_t_gd folder.
The cellranger vdj pipeline cannot process FASTQs from TRG/D enriched libraries.
10x Genomics does not support the use of cellranger aggr to aggregate the outputs of TRG/D enriched libraries.
Next, you may wish to:
- Understand multi outputs.
- Run cellranger aggr to aggregate the outputs from multiple runs of
cellranger multiand perform analysis on the combined data. - Explore Loupe and Loupe VDJ browser to visualize your data.