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10x Genomics
Chromium Single Cell ATAC

Direct Demultiplexing with Illumina Software


There are three options for generating FASTQ files from BCL files, all of which work for 10x Genomics Chromium libraries:

Illumina's software may provide greater control over demultiplexing parameters.

Demultiplexing Chromium data with Illumina's BCL Convert or bcl2fastq software requires the correct specification of the sample sheet and command line options. This guide walks you through generating Cell Ranger-compatible FASTQs with BCL Convert and bcl2fastq.

The ATAC library is single-indexed. You can download the Sample Index Kit N Set A as a CSV or JSON.

There is a key difference to keep in mind when creating single index sample sheets for a Chromium run. Each Chromium sample index set is a blend of four different sequence oligos, and each oligo must be represented as a separate row in the sample sheet. This means that for every sample being demultiplexed from the flow cell, there should be four lines in the sample sheet.

Select a tab for information about running bcl2fastq or bcl-convert:

Demultiplexing with bcl2fastq

Download and install bcl2fastq

The bcl2fastq software is available for download and installation on the Illumina support website as an RPM package. An Illumina account is required for download. Please contact Illumina Support if you have questions about bcl2fastq versions, or for help troubleshooting its download and installation. See 10x Genomics Knowledge Base article, How to troubleshoot installing bcl2fastq or bcl-convert?

Creating the sample sheet

You must create a sample sheet for bcl2fastq to correctly embed the names of samples into output FASTQ files. This section has an example single index ATAC sample sheet that can be customized for your experiment.

The Illumina Experiment Manager can also be used to create sample sheets for bcl2fastq.

Please note that the index sequence in the sample index reference file should be entered into the index column of the bcl2fastq sample sheet. Either the index2_workflow_a or index2_workflow_b sequence should be entered into the index2 column of the bcl2fastq sample sheet, depending on the sequencing instrument in use.

  • index2_workflow_a: NovaSeq™ 6000 v1, MiSeq™, HiSeq™ 2500, and HiSeq™ 2000.
  • index2_workflow_b: NovaSeq™ 6000 v1.5, iSeq™ 100, MiniSeq™, NextSeq™, HiSeq™ X, and HiSeq™ 3000/4000.

Example sample sheet

For each sample, enter its lane, sample name, and set of four sample indices into the Illumina bcl2fastq sample sheet. This sample sheet shows two samples, sample1 is split on two lanes (1 and 2) and the sample2 is only found on lane 1:


Running bcl2fastq

Illumina bcl2fastq must be called with the correct --use-bases-mask argument and other arguments to properly demultiplex and output FASTQs for all the reads in a Chromium library. Learn more about the --use-bases-mask argument in this Knowledge Base article.

In the examples below, ${FLOWCELL_DIR} is the directory that contains a flow cell's Data folder, ${OUTPUT_DIR} is the directory that you want to output FASTQs to, and ${SAMPLE_SHEET_PATH} is the path to the sample sheet CSV you created.

bcl2fastq v2.20 or later

 bcl2fastq --use-bases-mask=Y50,I8,Y24,Y50 \
  --create-fastq-for-index-reads \
  --minimum-trimmed-read-length=8 \
  --mask-short-adapter-reads=8 \
  --ignore-missing-positions \
  --ignore-missing-controls \
  --ignore-missing-filter \
  --ignore-missing-bcls \
  -r 6 -w 6 \
  --output-dir=${OUTPUT_DIR} \
  --interop-dir=${INTEROP_DIR} \

To limit bcl2fastq to a subset of lanes, supply values to the --tiles argument.

Omitting extra bases from reads

If you add extra bases to a sample index read, you will need to account for this in the --use-bases-mask argument. For example, if you ran a sample index read with 9 bases, you must truncate the last base for Cell Ranger ATAC to run correctly.

You can exclude a single base by adding a single n character to the read argument, or adding n* to exclude all bases after a certain position. See below:

i7 Index Read (I1)89I8n

Learn more about the --use-bases-mask argument in this Knowledge Base article.

Demultiplexing with BCL Convert

Illumina's BCL Convert is another software application that converts BCL files into FASTQ files. This page explains how to use BCL Convert for Chromium Single Cell ATAC libraries and provide an example sample sheet to use as input. In addition, there is a step-by-step guide with an example BCL dataset for generating FASTQs compatible with Cell Ranger ATAC.

Download and install BCL Convert

BCL Convert is available for download and installation on the Illumina support website as an RPM package. An Illumina account is required for download. Please contact Illumina Support if you have questions about BCL Convert versions, or need help troubleshooting its download and installation. See 10x Genomics Knowledge Base article, How to troubleshoot installing bcl2fastq or bcl-convert?

Creating the sample sheet

BCL Convert uses a sample sheet CSV file to specify sample information and parameters for a run, instead of command line options. For a full description of the sample sheet and list of settings, please see the Illumina documentation.

The basic sample sheet has three sections. Each section is described here and example sample sheets are provided for both single and dual indexed samples.

  • [Header] can be used to specify the BCL sample sheet version.

  • [BCLConvert_Settings] in a V2 sample sheet, this section is used to specify several FASTQ conversion settings including whether or not to create FASTQ files for indices. Use [Settings] in a V1 sample sheet. Leave adapter trim settings blank in this section.

  • [BCLConvert_Data] in a V2 sample sheet, this section is used to sort samples and index adapters based on the following column headers. The [BCLConvert_Data] section must be renamed [Data] or [data] for a V1 sample sheet:

    Column name Description
    Lane Optional. Generates FASTQ files only for the samples with the specified lane number. Allows only one valid integer. If the same sample has been run on multiple lanes of the flow cell, add a new row for each lane. If the lane is not specified, indices are searched in all lanes.
    Sample_ID The sample ID
    index I7 index sequence
    index2 I5 index sequence
    Sample_Project Optional. Used when --bcl-sampleproject-subdirectories is specified in BCL Convert run. Only alphanumeric characters, dashes, and underscores are allowed. Logs or Reports should not be used as directory names for this flag, as they are already default output directories. Learn more.

Single index ATAC library example

This section shows an example sample sheet for ATAC libraries created with the Single Index Kit N Set A. Please note that some settings under the [BCLConvert_Settings] section are different because ATAC libraries have a different read structure. The parameter CreateFastqForIndexReads,1 in combination with TrimUMI,0 tells BCL Convert to output UMI cycles to FASTQ files. The OverrideCycles parameter specifies the sequencing and indexing cycles that should be used when processing the data and must have the same number of semicolon-delimited fields in string as the sequencing and indexing reads specified in your RunInfo.xml.

This sample sheet shows two samples, sample1 and sample2. Since lane is not specified in this example, indices are searched in all lanes.


Running BCL Convert

The command to run BCL Convert:

bcl-convert --bcl-input-directory <folder-with-bcls> \
 --output-directory <name-of-output-dir-for-FASTQs> \
 --sample-sheet <samplesheet-filename.csv>

Required arguments:

  • --bcl-input-directory: path to the input directory containing BCL files
  • --output-directory: path to an output directory for newly created FASTQ files. This directory must not exist before command execution.
  • --sample-sheet: path to a CSV file containing sample information as described in the creating the sample sheet section. Providing a path to the directory instead of the specific CSV file can cause the software to hang.

Output FASTQs

A successful BCL Convert run looks like this:

Sample sheet being processed by common lib? Yes
SampleSheet Settings:
  CreateFastqForIndexReads = 1
  OverrideCycles = Y50;I8;U24;Y49
  TrimUMI = 0

shared-thread-linux-native-asio output is disabled
bcl-convert Version
Copyright (c) 2014-2022 Illumina, Inc.
Command Line: --bcl-input-directory miseq-atac --output-directory atac-fastqs --sample-sheet atac_samplesheet.
Conversion Begins.
# CPU hw threads available: 64
Parallel Tiles: 4. Threads Per Tile: 16
SW compressors: 64
SW decompressors: 32
SW FASTQ compression level: 1
Conversion Complete.

A new folder is created (name specified by the --output-directory flag). This folder contains FASTQ file sets, one per sample. The folder also contains Logs and Reports sub-directories that contain the run logs and metrics output files respectively.

Example dataset

A convenient way to test BCL Convert is by downloading the tiny bcl example dataset. This dataset has been selected for its relatively small size. The example below applies to Chromium Single Cell ATAC libraries. It should not be used to run downstream pipelines (e.g. cellranger-atac count).

To follow along:

tar -xvf /working-directory/cellranger-arc-tiny-bcl-atac-2.0.0.tar.gz

A folder called miseq-atac will appear in your working directory.

Run BCL Convert:

bcl-convert --bcl-input-directory /working-directory/miseq-atac \
--output-directory /working-directory/atac-fastqs \
--sample-sheet /working-directory/cellranger-arc-tiny-bcl-atac-samplesheet-2.0.0.csv

Remember to customize the --bcl-input-directory path with the path to your input directory. This command takes ~10 minutes to complete.

When the run is complete, a folder called atac-fastqs is created in the working directory. This folder contains your newly created FASTQ files.

├── atac-fastqs
│   ├── Logs
│   │   ├── Errors.log
│   │   ├── FastqComplete.txt
│   │   ├── Info.log
│   │   └── Warnings.log
│   ├── Reports
│   │   ├── Adapter_Cycle_Metrics.csv
│   │   ├── Adapter_Metrics.csv
│   │   ├── Demultiplex_Stats.csv
│   │   ├── Demultiplex_Tile_Stats.csv
│   │   ├── fastq_list.csv
│   │   ├── Index_Hopping_Counts.csv
│   │   ├── IndexMetricsOut.bin
│   │   ├── Quality_Metrics.csv
│   │   ├── Quality_Tile_Metrics.csv
│   │   ├── RunInfo.xml
│   │   ├── SampleSheet.csv
│   │   └── Top_Unknown_Barcodes.csv
│   ├── test_sample_S1_L001_I1_001.fastq.gz
│   ├── test_sample_S1_L001_I2_001.fastq.gz
│   ├── test_sample_S1_L001_R1_001.fastq.gz
│   ├── test_sample_S1_L001_R2_001.fastq.gz
│   ├── Undetermined_S0_L001_I1_001.fastq.gz
│   ├── Undetermined_S0_L001_I2_001.fastq.gz
│   ├── Undetermined_S0_L001_R1_001.fastq.gz
│   └── Undetermined_S0_L001_R2_001.fastq.gz
├── bcl_convert_samplesheet.csv
└── runinfo.txt

Illumina support and documentation

Please contact Illumina Support for general questions about BCL Convert. Or refer to BCL Convert documentation:

Next steps

After generating FASTQs, you are ready to run the cellranger-atac count.