10x Genomics
Chromium Single Cell Multiome ATAC + Gene Exp.

Cell Ranger ARC1.0, printed on 11/05/2024

Single-Library Analysis with Cell Ranger ARC

Cell Ranger ARC's pipelines analyze sequencing data produced from Chromium Single Cell Multiome ATAC + Gene Expression. The analysis involves the following steps:

1. Run cellranger-arc mkfastq on the Illumina BCL output folder for each ATAC (GEX resp.) flow cell to generate ATAC (GEX resp.) FASTQ data. Note that a separate run of mkfastq is required for each ATAC and each GEX flow cell.

2. Run a separate instance of cellranger-arc count for each GEM well that was demultiplexed by the cellranger-arc mkfastq in the previous step.

For the following example, assume that one sample is processed using Single Cell Multiome ATAC + Gene Expression to generate a Multiome ATAC library and a Multiome Gene Expression (GEX) library. The Multiome ATAC library is sequenced on flow cell HNATACSQXX and the Illumina BCL output is located in /sequencing/Sample_ATAC_HNATACSQXX; similarly, the Multiome GEX library is sequenced on flow cell HNGEXSQXXX and the Illumina BCL output is located in /sequencing/Sample_GEX_HNGEXSQXXX.

Run cellranger-arc mkfastq

Follow the instructions on running cellranger-arc mkfastq to generate FASTQ files for both the ATAC and GEX flow cells. cellranger-arc mkfastq will create output ATAC FASTQ files in HNATACSQXX/outs/fastq_path and GEX FASTQ files in HNGEXSQXXX/outs/fastq_path.

Download or create a cellranger-arc-compatible reference

Reference packages for human (GRCh38) and mouse (mm10) compatible with Cell Ranger ARC are available for download. You can also create a reference package using cellranger-arc mkref starting with a genome assembly FASTA file, a GTF file of gene annotations, and optionally a file of transcription factor motifs in JASPAR format.

Create a libraries CSV file

Construct a 3-column libraries CSV file that specifies the location of the ATAC and GEX FASTQ files associated with the sample.

For our example, the file would look as follows:

fastqs,sample,library_type
/home/jdoe/runs/HNGEXSQXXX/outs/fastq_path,example,Gene Expression
/home/jdoe/runs/HNATACSQXX/outs/fastq_path,example,Chromatin Accessibility

The CSV contains two rows as all the sequencing data for ATAC (GEX resp.) were obtained from one flow cell. The library_type is restricted to be either Gene Expression or Chromatin Accessibility.

Run cellranger-arc count

To generate single cell feature counts and secondary analyses for a single library, run cellranger-arc count with the following arguments. For a complete listing of the arguments accepted, see the Command Line Argument Reference below, or run cellranger-arc count --help.

After determining these input arguments, run cellranger-arc:

$ cd /home/jdoe/runs
$ cellranger-arc count --id=sample345 \
                       --reference=/opt/refdata-cellranger-arc-GRCh38-2020-A \
                       --libraries=/home/jdoe/runs/libraries.csv \
                       --localcores=16 \
                       --localmem=64

Following a series of checks to validate input arguments, cellranger-arc count pipeline stages will begin to run:

Martian Runtime - v4.0.1
 

Running preflight checks (please wait)...
Checking FASTQ folder...
Checking reference...
Checking reference_path (/opt/refdata-cellranger-arc-GRCh38-2020-A) on compute-server32...
Checking chemistry...
Checking optional arguments...
...

By default, cellranger-arc will use all 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 cellranger-arc to using up to sixteen cores at once. Similarly, --localmem will restrict the amount of memory (in GB) used by cellranger-arc.

The pipeline will create a new folder named with the sample ID you specified (e.g. /home/jdoe/runs/sample345) for its output. If this folder already exists, cellranger-arc will assume it is an existing pipestance and attempt to resume running it.

Output Files

A successful cellranger-arc count run should conclude with a message similar to this:

Outputs:
- Secondary analysis outputs:
    clustering:
      atac: {
        ...
      }
      gex:  {
        ...
      }
    dimensionality_reduction:
      atac: {
        ...
      }
      gex:  {
        ...
      }
    feature_linkage:
      ...
    tf_analysis:
      ...
- Run summary HTML:                              /home/jdoe/runs/sample345/outs/web_summary.html
- Run summary metrics CSV:                       /home/jdoe/runs/sample345/outs/summary.csv
- Per barcode summary metrics:                   /home/jdoe/runs/sample345/outs/per_barcode_metrics.csv
- Filtered feature barcode matrix MEX:           /home/jdoe/runs/sample345/outs/filtered_feature_bc_matrix
- Filtered feature barcode matrix HDF5:          /home/jdoe/runs/sample345/outs/filtered_feature_bc_matrix.h5
- Raw feature barcode matrix MEX:                /home/jdoe/runs/sample345/outs/raw_feature_bc_matrix
- Raw feature barcode matrix HDF5:               /home/jdoe/runs/sample345/outs/raw_feature_bc_matrix.h5
- Loupe browser visualization file:              /home/jdoe/runs/sample345/outs/cloupe.cloupe
- GEX Position-sorted alignments BAM:            /home/jdoe/runs/sample345/outs/gex_possorted_bam.bam
- GEX Position-sorted alignments BAM index:      /home/jdoe/runs/sample345/outs/gex_possorted_bam.bam.bai
- GEX Per molecule information file:             /home/jdoe/runs/sample345/outs/gex_molecule_info.h5
- ATAC Position-sorted alignments BAM:           /home/jdoe/runs/sample345/outs/atac_possorted_bam.bam
- ATAC Position-sorted alignments BAM index:     /home/jdoe/runs/sample345/outs/atac_possorted_bam.bam.bai
- ATAC Per fragment information file:            /home/jdoe/runs/sample345/outs/atac_fragments.tsv.gz
- ATAC Per fragment information index:           /home/jdoe/runs/sample345/outs/atac_fragments.tsv.gz.tbi
- ATAC peak locations:                           /home/jdoe/runs/sample345/outs/atac_peaks.bed
- ATAC smoothed transposition site track:        /home/jdoe/runs/sample345/outs/atac_cut_sites.bigwig
- ATAC peak annotations based on proximal genes: /home/jdoe/runs/sample345/outs/atac_peak_annotation.tsv
 
Waiting 6 seconds for UI to do final refresh.
Pipestance completed successfully!
 
yyyy-mm-dd hh:mm:ss Shutting down.
 
Saving pipestance info to "sample345/sample345.mri.tgz"

The output of the pipeline will be contained in a folder named with the sample ID you specified (e.g. sample345). The subfolder named outs will contain the main pipeline output files:

Once cellranger-arc count has successfully completed, you can browse the resulting summary HTML file in any supported web browser, open the .cloupe file in Loupe Browser, or refer to the Understanding Output section to explore the data manually.

Command-Line Argument Reference

This table is a partial list of commonly used command-line arguments. The full list of options can be obtained via cellranger-arc count --help.