10x Genomics Support/Cell Ranger/Tutorials/

Analyzing Barcode Enabled Antigen Capture for T Cells (BEAM-T) with Cell Ranger multi

This tutorial is written with Cell Ranger v7.1.0. Starting with Cell Ranger v8.0, it is mandatory to use the --create-bam parameter when executing the cellranger count and cellranger multi pipelines. This new parameter replaces the previously used --no-bam option. All other arguments remain compatible with newer versions, unless otherwise specified.

To follow along, you must:

  • Have basic UNIX command line experience
  • Fulfill these system requirements
  • Download and install the Cell Ranger software
  • Choose a compute platform
  • Have access to a UNIX command prompt

We will work with the 5k Human A0201 | B0702 PBMCs (BEAM-T) dataset.

Open up a terminal window. You may log in to a remote server or choose to perform the compute on your local machine. Refer to the System Requirements page for details.

In the working directory, create a new folder called beam-t and cd into that folder:

mkdir beam-t cd beam-t

Download the input FASTQ files:

curl -O https://cf.10xgenomics.com/samples/cell-vdj/7.1.0/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_fastqs.tar

A file named 5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_fastqs.tar should appear in your directory when you list files with the ls -lt command.

Decompress the FASTQs:

tar -xf 5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_fastqs.tar

You should now see a folder called 5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_fastq

cd 5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_fastq ls

The folder contains three subfolders with library-specific FASTQS files: antigen_capture, gex, and vdj.

Navigate back to the working directory:

cd ..

Double check you are in the correct directory by running the ls command; the working directory should have the FASTQs 5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_fastqs folder.

Download the Feature Reference CSV available for this example dataset.

curl -O https://cf.10xgenomics.com/samples/cell-vdj/7.1.0/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_count_feature_reference.csv

To view the contents of the Feature Reference CSV, open it in your text editor of choice (e.g., nano)

nano 5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_count_feature_reference.csv

The contents should look like this:

id,name,read,pattern,sequence,feature_type,mhc_allele Flu_A0201,Flu_A0201,R2,^(BC),GATTGGCTACTCAAT,Antigen Capture,HLA-A*02:01 CMV_B0702,CMV_B0702,R2,^(BC),CGGCTCACCGCGTCT,Antigen Capture,HLA-B*07:02 negative_control_A0201,negative_control_A0201,R2,^(BC),CTATCTACCGGCTCG,Antigen Capture,HLA-A*02:01 negative_control_B0702,negative_control_B0702,R2,^(BC),CATGTCTACGTTAAG,Antigen Capture,HLA-B*07:02

Since this is a BEAM-T (TCR Antigen Capture) dataset, the Feature Reference CSV contains the additional mhc_allele column. The BEAM-Ab tutorial guides you through analyzing a BCR Antigen Capture dataset.

When working with your own dataset, you must customize this file for your experiment. Learn more about the Feature Reference CSV.

As of this tutorial's publication, the most current is the Human reference (GRCh38) - 2020-A. Download the pre-built human reference transcriptome to the working directory (beam-t) and decompress it:

curl -O https://cf.10xgenomics.com/supp/cell-exp/refdata-gex-GRCh38-2020-A.tar.gz tar -xf refdata-gex-GRCh38-2020-A.tar.gz

Next, download the pre-built V(D)J reference to the working directory and uncompress it:

curl -O https://cf.10xgenomics.com/supp/cell-vdj/refdata-cellranger-vdj-GRCh38-alts-ensembl-7.1.0.tar.gz tar -xf refdata-cellranger-vdj-GRCh38-alts-ensembl-7.1.0.tar.gz

In your working directory, create a new CSV file called 5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_config.csv using your text editor of choice. For example, you can create a file with nano using this command:

nano 5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_config.csv

Copy and paste this text into the newly created file and customize the /path/to/... part of file paths:

[gene-expression] ref,/path/to/references/refdata-gex-GRCh38-2020-A create-bam,true [feature] ref,/path/to/feature_references/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_count_feature_reference.csv [vdj] ref,/path/to/references/vdj/refdata-cellranger-vdj-GRCh38-alts-ensembl-7.1.0 [libraries] fastq_id,fastqs,lanes,feature_types beamt_human_A0201_B0702_pbmc_ag,/path/to/fastqs/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_fastqs/antigen_capture,1|2,Antigen Capture beamt_human_A0201_B0702_pbmc_vdj,/path/to/fastqs/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_fastqs/vdj,1|2,VDJ-T beamt_human_A0201_B0702_pbmc_gex,/path/to/fastqs/5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_fastqs/gex,1|2,Gene Expression [antigen-specificity] control_id,mhc_allele negative_control_A0201,HLA-A*02:01 negative_control_B0702,HLA-B*07:02

Use your text editor's save command to save the file. In nano, save by typing CTRL+XyENTER.

A customizable multi config CSV template is available for download on the example dataset page, under the Input Files tab.

Once you have all the necessary files, make a new directory called runs/ in your beam-t/ working directory:

mkdir runs/ cd runs/

You will run cellranger multi in the runs/ directory.

After downloading/creating the FASTQ files, Feature Reference CSV, reference transcriptome, and V(D)J reference, you are ready to run cellranger multi.

Print the usage statement to get a list of all the options:

cellranger multi --help

The output should look similar to:

user_prompt$ cellranger multi --help cellranger-multi Analyze multiplexed data or combined gene expression/immune profiling/feature barcode data USAGE: cellranger multi [FLAGS] [OPTIONS] --id --csv FLAGS: --dry Do not execute the pipeline. Generate a pipeline invocation (.mro) file and stop --disable-ui Do not serve the web UI --noexit Keep web UI running after pipestance completes or fails --nopreflight Skip preflight checks -h, --help Prints help information OPTIONS: --id A unique run id and output folder name [a-zA-Z0- 9_-]+ --description Sample description to embed in output files [default: ] --csv Path of CSV file enumerating input libraries and analysis parameters --jobmode Job manager to use. Valid options: local (default), sge, lsf, slurm or path to a .template file. Search for help on "Cluster Mode" at support.10xgenomics.com for more details on configuring the pipeline to use a compute cluster [default: local] --localcores Set max cores the pipeline may request at one time. Only applies to local jobs ....

Options used in this tutorial

--idThe id argument must be a unique run ID. We will call this run HumanB_Cell_multi based on the sample type in the example dataset.
--csvPath to the multi config CSV file enumerating input libraries and analysis parameters. Your multi_config.csv file is in the working directory. When executing cellranger multi from the runs directory, the relative path should be: ../multi_config.csv

From within the beam-t/runs/ directory, run cellranger multi

/path/to/cellranger-7.1.0/cellranger multi --id=beam-t-run --csv=../5k_BEAM-T_Human_A0201_B0702_PBMC_5pv2_Multiplex_config.csv

The run begins similarly to this:

user_prompt$ cellranger multi --id=beam-t-run --csv=/jane.doe/beam-t/multi_config.csv Martian Runtime - v4.0.10 2023-06-15 11:44:24 [jobmngr] WARNING: configured to use 334GB of local memory, but only 194.9GB is currently available. Serving UI at http://bespin3.fuzzplex.com:34513?auth=-Sm5gsg6_G8FjcUX0_YD5J8SYoBODz4IWoVIK9ec0jg Running preflight checks (please wait)... 2023-06-15 11:44:33 [runtime] (ready) ID.beam-t-run.SC_MULTI_CS.PARSE_MULTI_CONFIG 2023-06-15 11:44:33 [runtime] (run:local) ID.beam-t-run.SC_MULTI_CS.PARSE_MULTI_CONFIG.fork0.chnk0.main 2023-06-15 11:44:56 [runtime] (chunks_complete) ID.beam-t-run.SC_MULTI_CS.PARSE_MULTI_CONFIG 2023-06-15 11:44:56 [runtime] (ready) ID.beam-t-run.SC_MULTI_CS.FULL_COUNT_INPUTS.WRITE_GENE_INDEX 2023-06-15 11:44:56 [runtime] (run:local) ID.beam-t-run.SC_MULTI_CS.FULL_COUNT_INPUTS.WRITE_GENE_INDEX.fork0.chnk0.main ....

When the output of the cellranger multi command says, “Pipestance completed successfully!”, the job is done:

web_summary: /jane.doe/beam-t/runs/beam-t-run/outs/per_sample_outs/beam-t/web_summary.html metrics_summary: /jane.doe/beam-t/runs/beam-t-run/runs/beam-t/outs/per_sample_outs/beam-t/metrics_summary$ } Waiting 6 seconds for UI to do final refresh. Pipestance completed successfully!

A successful cellranger multi run produces a new directory called beam-t-run (based on the --id flag specified during the run). The contents of the beam-t-run/ directory:

. ├── beam-t-run │ ├── beam-t.mri.tgz │ ├── _cmdline │ ├── _filelist │ ├── _finalstate │ ├── _invocation │ ├── _jobmode │ ├── _log │ ├── _mrosource │ ├── outs │ ├── _perf │ ├── _perf._truncated_ │ ├── SC_MULTI_CS │ ├── _sitecheck │ ├── _tags │ ├── _timestamp │ ├── _uuid │ ├── _vdrkill │ └── _versions

The outs/ directory contains all important output files generated by the cellranger multi pipeline:

── runs └── beam-t-run └──outs ├── config.csv ├── multi │ ├── count │ │ ├── feature_reference.csv │ │ ├── raw_cloupe.cloupe │ ├── raw_feature_bc_matrix │ │ ├── raw_feature_bc_matrix.h5 │ │ ├── raw_molecule_info.h5 │ │ ├── unassigned_alignments.bam │ │ └── unassigned_alignments.bam.bai │ └── vdj_t │ ├── all_contig_annotations.bed │ ├── all_contig_annotations.csv │ ├── all_contig_annotations.json │ ├── all_contig.bam │ ├── all_contig.bam.bai │ ├── all_contig.fasta │ ├── all_contig.fasta.fai │ └── all_contig.fastq ├── per_sample_outs │ └── beam-t │ ├── antigen_analysis │ ├── count │ ├── metrics_summary.csv │ ├── vdj_t │ └── web_summary.html └── vdj_reference ├── fasta │ ├── donor_regions.fa │ └── regions.fa └── reference.json