10x Genomics
Chromium Single Cell Immune Profiling

Cell Ranger4.0, printed on 11/05/2024

Analysis of Multiple Library Types

Single Cell V(D)J + 5′ Gene Expression + Feature Barcode Technology for Cell Surface Protein Analysis

The Chromium Single Cell Immune Profiling Solution with Feature Barcode technology enables analysis of V(D)J clonotypes, gene expression, cell surface protein, and antigen specificity (Feature Barcode) simultaneously, in the same single cell.

Analysis of each of the sequenced libraries, alone or in combination with other library types, can be performed using the latest versions of Cell Ranger, Loupe Browser, and Loupe V(D)J Browser.

Running Cell Ranger

Assume a sample sheet was used that yielded FASTQs with sample name prefixes SampleGEX, SampleFB, SampleT and SampleB. To process the four different library types, run separate instances of the Cell Ranger pipelines:

• cellranger vdj to analyze data from the SampleT and SampleB libraries separately. Two instances of cellranger vdj need to be run for SampleT and SampleB.
• cellranger count to analyze gene expression from the SampleGEX library. Count pipeline also performs Feature Barcode analysis simultaneously with Gene Expression analysis. Consult Cell Ranger Feature Barcode Analysis in the Gene Expression section for more details.

To analyze the data from the enriched T cell or B cell libraries, run the cellranger vdj pipeline:

$ cd /home/jdoe/runs
$ cellranger vdj --id=GEX_VDJ_SampleT \
                 --reference=/opt/refdata-cellranger-vdj-GRCh38-alts-ensembl-4.0.0 \
                 --fastqs=/home/jdoe/mkfastq_pipeline/outs/fastq_path \
                 --sample=SampleT \

$ cd /home/jdoe/runs
$ cellranger vdj --id=GEX_VDJ_SampleB \
                 --reference=/opt/refdata-cellranger-vdj-GRCh38-alts-ensembl-4.0.0 \
                 --fastqs=/home/jdoe/mkfastq_pipeline/outs/fastq_path \
                 --sample=SampleB \

These pipelines generate a Loupe V(D)J Browser .vloupe file, in addition to a variety of files to describe V(D)J clonotypes, consensus sequences, and contigs. For more details on running the V(D)J analysis pipeline, consult the Cell Ranger V(D)J documentation.

To analyze the data from the gene expression and Feature Barcode library, run the cellranger count pipeline:

$ cd /home/jdoe/runs
$ cellranger count --id=GEX_VDJ_SampleGEX \
                   --transcriptome=/opt/refdata-gex-GRCh38-2020-A \
                   --libraries=libraries.csv \
                   --feature-ref=feature-ref.csv \

This generates a Loupe Browser .cloupe file, a feature-barcode matrix, and other information. For more details on running the cellranger count pipeline, consult the Cell Ranger Gene Expression documentation and Cell Ranger Feature Barcode Analysis in the Gene Expression section for more details.

Loupe Tutorials

The Loupe Browser and Loupe V(D)J Browser allow you to tie gene expression, cell surface protein and V(D)J information from the three pipeline runs, back together.

To learn more about how to conduct multimodal analysis of V(D)J and gene expression data from the same sample, start the Loupe V(D)J + Gene Expression Tutorial. In the tutorial, you will be able to download example 5′ gene expression profiles and T cell clonotypes from a non-small cell lung carcinoma, and use the Loupe browsers to analyze the integrated data.

To learn more about analyzing Feature Barcode data, start the Immune Profile Analysis with Feature Barcode and Surface Marker Expression tutorial. In this tutorial, you will download and explore 5′ gene expression and Cell Surface Protein data from a PBMC sample with Loupe Browser.