The cellranger-atac workflow starts by demultiplexing the Illumina sequencer's base call files (BCLs) for each flow cell directory into FASTQ files. 10x Genomics recommends using cellranger-atac mkfastq, a pipeline that wraps Illumina's bcl2fastq software and provides a number of convenient features in addition to the features of bcl2fastq:

• Translates 10x Genomics sample index set names into the corresponding list of four sample index oligonucleotides. For example, well A1 can be specified in the sample sheet as SI-NA-A1, and cellranger-atac mkfastq will recognize the four oligos (AAACGGCG, CCTACCAT, GGCGTTTC, and TTGTAAGA) and merge the resulting FASTQ files.
• Supports a simplified CSV sample sheet format to handle 10x Genomics use cases.
• Supports most bcl2fastq arguments, such as --use-bases-mask.

The compute workflow begins with running one instance of cellranger-atac mkfastq for each flow cell of data being analyzed. Once the data are successfully demultiplexed, one instance of cellranger-atac count is run for each Epi ATAC library, independent of the number of sequencing runs of each library.

In the first example, two 10x Genomics libraries (each processed through a separate Chromium chip channel) are multiplexed on a single flow cell. After running cellranger-atac mkfastq, run a separate instance of the cellranger-atac count pipeline on each library:

In the second example, one 10x Genomics library was sequenced on two flow cells. After running cellranger-atac mkfastq, run a single instance of the cellranger-atac count pipeline on all the FASTQ files generated:

The cellranger-atac mkfastq pipeline accepts additional options beyond those shown in the table below because it is a wrapper around bcl2fastq. Consult the User Guide for Illumina's bcl2fastq for more information.

The cellranger-atac mkfastq pipeline recognizes two file formats for describing samples: a simple, three-column CSV format, or the Illumina Experiment Manager (IEM) sample sheet format used by bcl2fastq. Both of these formats are illustrated below.

To follow along:

1. Download the tiny-bcl-atac tar file.
2. Untar the tiny-bcl tar file in a convenient location. This will create a new tiny-bcl/ subdirectory.
3. Download the simple CSV layout file: cellranger-atac-tiny-bcl-simple-1.0.0.csv.
4. Download the Illumina Experiment Manager sample sheet: cellranger-atac-tiny-bcl-samplesheet-1.0.0.csv.

A simple CSV sample sheet is recommended for most sequencing experiments. The simple CSV format has only three columns (Lane, Sample, Index), and is thus less prone to formatting errors. You can see an example of this in cellranger-atac-tiny-bcl-simple-1.0.0.csv:

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Lane,Sample,Index
1,test_sample_atac,SI-NA-C1

Here are the options for each column:

To run cellranger-atac mkfastq with a simple layout CSV, use the --csv argument. Here's how to run cellranger-atac mkfastq on the tiny-bcl-atac sequencing run with the simple layout (replace /path/to/tiny_bcl with the path to tiny-bcl on your system):

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$ cellranger-atac mkfastq --id=tiny-bcl \
                     --run=/path/to/tiny_bcl \
                     --csv=cellranger-atac-tiny-bcl-simple-1.0.0.csv

The cellranger-atac mkfastq pipeline can also be run with a sample sheet in the Illumina Experiment Manager (IEM) format. An IEM sample sheet has several fields specific to running on Illumina platforms, including a [Data] section where sample and index information is specified. cellranger-atac mkfastq supports listing either index set names or the oligo sequences.

For example, "SI-NA-C1" refers to a 10x Genomics single-indexed sample index consisting of a set of four oligo sequences. In this example, only reads from lane 1 will be used. To demultiplex the given sample index across all lanes, omit the lanes column entirely.

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[Data]
Lane,Sample_ID,index
1,test_sample,SI-NA-C1

In this example, the four index sequences for "SI-NA-C1" are specified in separate rows under the index column.

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[Data]
Lane,Sample_ID,index
1,sample1,ATCTGATC
1,sample1,CGTGCTAA
1,sample1,GAGAAGGG
1,sample1,TCACTCCT

Sample names must conform to the Illumina bcl2fastq naming requirements. Only letters, numbers, underscores, and hyphens are allowed. No other symbols, including dots ("."), are allowed.

Also note that while an authentic IEM sample sheet will contain other sections above the [Data] section, these are optional for demultiplexing. To avoid data loss from trimming, we do not recommend including adapter sequences in the [Settings] section of the sample sheet. For demultiplexing an existing run with cellranger-atac mkfastq, only the [Data] section is required.

Next, run the cellranger-atac mkfastq pipeline, using the --samplesheet argument (replace /path/to/tiny-bcl-atac with the path to tiny_bcl on your system):

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$ cellranger-atac mkfastq --id=tiny-bcl \
 --run=/path/to/tiny_bcl \
 --samplesheet=cellranger-atac-tiny-bcl-samplesheet-1.0.0.csv

If you encounter any preflight errors, refer to the Troubleshooting page.

Once the cellranger-atac mkfastq pipeline has successfully completed, the output can be found in a new folder named with the value provided to cellranger-atac mkfastq in the --id option (if not specified, defaults to the name of the flow cell):

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$ ls -l
drwxrwxr-x 4 jdoe jdoe 4096 Aug 29 15:29 tiny-bcl-atac

The key output files can be found in outs/fastq_path, and are organized in the same manner as a conventional bcl2fastq run:

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$ ls -l tiny-bcl/outs/fastq_path/
drwxr-xr-x 3 jdoe jdoe 3 Aug 9 12:26 Reports
drwxr-xr-x 2 jdoe jdoe 8 Aug 9 12:26 Stats
drwxr-xr-x 3 jdoe jdoe 3 Aug 9 12:26 tiny-bcl
-rw-r--r-- 1 jdoe jdoe 20615106 Aug 9 12:26 Undetermined_S0_L001_I1_001.fastq.gz
-rw-r--r-- 1 jdoe jdoe 151499694 Aug 9 12:26 Undetermined_S0_L001_R1_001.fastq.gz
-rw-r--r-- 1 jdoe jdoe 52692701 Aug 9 12:26 Undetermined_S0_L001_R2_001.fastq.gz
-rw-r--r-- 1 jdoe jdoe 151499694 Aug 9 12:26 Undetermined_S0_L001_R3_001.fastq.gz

$ tree tiny-bcl/outs/fastq_path/tiny_bcl/
tiny-bcl/outs/fastq_path/tiny_bcl/
Sample1
Sample1_S1_L001_I1_001.fastq.gz
Sample1_S1_L001_R1_001.fastq.gz
Sample1_S1_L001_R2_001.fastq.gz
Sample1_S1_L001_R3_001.fastq.gz

This example was produced with a sample sheet that included tiny-bcl as the Sample_Project, so the directory containing the sample folders is named tiny-bcl. If a Sample_Project was not specified, or if a simple layout CSV file was used (which does not have a Sample_Project column), the directory containing the sample folders would be named according to the flow cell ID instead.

To remove the Undetermined FASTQs from the output, you can run mkfastq with the --delete-undetermined flag. To see all cellranger-atac mkfastq options, run cellranger-atac mkfastq --help.

If the pipeline crashes while running cellranger-atac mkfastq, upload this tarball (with the extension .mri.tgz) found in your output directory. Replace your@email.edu with your email

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cellranger-atac upload your@email.edu jobid.mri.tgz

where jobid is what you input into the --id option of mkfastq (if not specified, defaults to the ID of the flow cell).

This tarball contains numerous diagnostic logs that we can use for debugging.

You will receive an automated email from 10x Genomics. If not, email support@10xgenomics.com. For the fastest service, respond with the following:

• The exact cellranger-atac command line you used.
• The sample sheet that you used.
• The RunInfo.xml and runParameters.xml files from your BCL directory.
• The kind of libraries you are demultiplexing (including chemistry).

• Run cellranger-atac count.
• Learn how to specify FASTQs: Input FASTQ files must conform to naming conventions to successfully complete.