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10x Genomics
Chromium Single Cell Immune Profiling

Analysis of Multiple Library Types

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

The Chromium Single Cell Immune Profiling Solution with Feature Barcoding technology enables analysis of gene expression, V(D)J clonotypes, and cell surface protein expression simultaneously, in the same single cell. See V(D)J and Feature Barcoding technology This makes the Immune Profiling Solution ideal for studying immune response inside complex tissue samples, for observing the effects of immunotherapies, and investigating complex autoimmune, infectious, and other immune diseases.

The Immune Profiling Solution user guides provide the complete workflows required to generate three distinct library types. The recommended sequencing configuration for all library types (alone or in combination) is 26 x 91 bp. The V(D)J Enriched libraries (alone or in combination with 5’ Gene Expression and/or Cell Surface Protein libraries) may also be sequenced at 150 x 150 bp. For additional information, consult the Single Cell Immune Profiling Sequencing page and the Experimental Design for VDJ Libraries page.

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 Cell Browser, and Loupe V(D)J Browser Click the links to download.

Integrated Analysis using Cell Ranger, Loupe Cell Browser, and Loupe V(D)J Browser

Three distinct library types can be generated using the Chromium Single Cell Immune Profiling Solution with Feature Barcoding technology. Each library type can be analyzed using the pipelines and browsers specified the the table below.

For analysis of two or more library types, the pipeline and browser specified for each of the library types should be used. For example, when analyzing the V(D)J, 5’ Gene Expression, and Cell Surface Protein libraries in combination, use the Cellranger V(D)J pipeline and the Loupe V(D)J browser for the V(D)J library; the Cellranger Count pipeline and the Loupe Cell Browser for 5’ Gene Expression and Cell Surface Protein libraries.

Running Cell Ranger

Assume a sample sheet was used that yielded FASTQs with sample name prefixes SampleGEX, SampleT and SampleB. To process the three different library types, run separate instances of the new Cell Ranger 3.0 pipelines:

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-3.1.0 \
                 --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-3.1.0 \
                 --fastqs=/home/jdoe/mkfastq_pipeline/outs/fastq_path \
                 --sample=SampleB \

These pipelines will 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 library, run the cellranger count pipeline:

$ cd /home/jdoe/runs
$ cellranger count --id=GEX_VDJ_SampleGEX \
                   --transcriptome=/opt/refdata-cellranger-GRCh38-1.2.0 \
                   --fastqs=/home/jdoe/mkfastq_pipeline/outs/fastq_path \
                   --sample=SampleGEX \

This will generate a Loupe Cell Browser .cloupe file, a gene-barcode matrix, and other information. For more details on running the cellranger count pipeline, consult the Cell Ranger Gene Expression documentation.

If you generated a Feature Barcoding library alongside the gene expression library, it must be processed at the same time as the gene expression library. Consult the Cell Ranger Feature Barcoding Analysis in the Gene Expression section for more details.

Loupe Analysis

The Loupe Cell Browser and Loupe V(D)J Browser software allow you to tie gene expression and V(D)J information from these three pipeline runs back together. In Loupe Cell Browser 2.0 and greater, you can import T Cell and B Cell V(D)J .vloupe files into the Loupe Cell Browser workspace, allowing you to explore the immune repertoire within individual clusters, measure the gene expression of cells within a clonotype of interest, and create new clusters based on V(D)J data:

Click here for more information about how to use Loupe Cell Browser for gene expression and V(D)J analysis.

Likewise, you can import gene expression .cloupe files into Loupe V(D)J Browser, in order to compare clonotypes between gene expression clusters, and explore V(D)J sequences within clusters in greater detail:

Click here for more information about how to use Loupe V(D)J Browser for more detailed analysis of V(D)J data.

Example Dataset and Tutorial

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 Barcoding data, start the Immune Profile Analysis with Feature Barcoding 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 Cell Browser.