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

• Cell Ranger mkfastq: a 10x Genomics wrapper for bcl2fastq
• Illumina: bcl2fastq
• Illumina: BCL Convert

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 Multiome GEX library is dual-indexed. This section describes how to configure bcl2fastq or bcl-convert for GEX libraries created with the Dual Index Plate TT, Set A.

The Dual Index Plate TT, Set A are 'unique dual-indexing' sample indexes. This means that there is a unique sample index barcode in both the i7 and i5 index reads (also known as I1 and I2 respectively). When demultiplexing flow cells where both index reads have been sequenced, bcl2fastq and bcl-convert require that both index sequences match the expected sequence for a read to be assigned to that sample. This solves the 'index hopping' issue present on Illumina patterned flow cell sequencers.

You can download the Sample Index Reference files for the Gene Expression dual indexing kits here: Dual Index Plate TT, Set A CSV or Dual Index Plate TT, Set A JSON.

The Multiome 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:

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 at techsupport@illumina.com 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?

You must create a sample sheet for bcl2fastq to correctly embed the names of samples into output FASTQ files. This section has an example dual index GEX sample sheet and 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.

More information about dual-indexing is available in the Illumina Indexed Sequencing Overview Guide

When you plan an experiment, you should know the name of the sample index set used for each sample, which comes from the reagent kit (such as "SI-TT-A2"). For each sample, enter its lane, sample name, and sample index set into the Illumina bcl2fastq sample sheet. Here is a bcl2fastq sample sheet for a HiSeq 2500. This sample sheet shows two samples, sample1 is split on two lanes (1 and 2) and the sample2 is only found on lane 1:

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[Data]
Lane,Sample_ID,index,index2
1,sample1,GTAACATGCG,AGGTAACACT
2,sample1,GTAACATGCG,AGGTAACACT
1,sample2,GTGGATCAAA,GCCAACCCTG

For each sample, enter its lane, sample name, and set of four sample indices into the Illumina bcl2fastq sample sheet. Here is an example using "SI-GA-A1" indices. This sample sheet shows two samples, sample1 is split on two lanes (1 and 2) and the sample2 is only found on lane 1:

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[Data]
Lane,Sample_ID,index
1,sample1,GGTTTACT
1,sample1,CTAAACGG
1,sample1,TCGGCGTC
1,sample1,AACCGTAA
2,sample1,GGTTTACT
2,sample1,CTAAACGG
2,sample1,TCGGCGTC
2,sample1,AACCGTAA
1,sample2,AAACGGCG
1,sample2,CCTACCAT
1,sample2,GGCGTTTC
1,sample2,TTGTAAGA

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

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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 \
  -R ${FLOWCELL_DIR} \
  --output-dir=${OUTPUT_DIR} \
  --interop-dir=${INTEROP_DIR} \
  --sample-sheet=${SAMPLE_SHEET_PATH}

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

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 ARC 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:

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

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 Multiome ATAC + Gene Expression libraries and provides example sample sheets to use as inputs. In addition, there is a step-by-step guide with an example BCL dataset for generating FASTQs compatible with Cell Ranger ARC.

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 at techsupport@illumina.com 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?

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.

This section shows an example sample sheet for GEX libraries created with the Dual Index Kit TT, Set A. The parameter CreateFastqForIndexReads,0 under [BCLConvert_Settings] tells BCL Convert not to generate FASTQ files for indices. Cell Ranger does not require FASTQ files for indices. This sample sheet shows two samples, sample1 is split on two lanes (1 and 2) and the sample2 is only found on lane 1:

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[Header]
FileFormatVersion,2

[BCLConvert_Settings]
CreateFastqForIndexReads,0

[BCLConvert_Data]
Lane,Sample_ID,index,index2
1,sample1,GTAACATGCG,AGGTAACACT
2,sample1,GTAACATGCG,AGGTAACACT
2,sample2,GTGGATCAAA,GCCAACCCTG

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.

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[Header]
FileFormatVersion,2

[BCLConvert_Settings]
CreateFastqForIndexReads,1
TrimUMI,0
OverrideCycles,Y50;I8;U24;Y49

[BCLConvert_Data]
Sample_ID,index
Sample1_SI,GGTTTACT
Sample1_SI,CTAAACGG
Sample1_SI,TCGGCGTC
Sample1_SI,AACCGTAA
Sample2_SI,AGCTGCGT
Sample2_SI,CAACCATC
Sample2_SI,GTGGAGCA
Sample2_SI,TCTATTAG

The command to run BCL Convert:

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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.

A successful BCL Convert run looks like this:

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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 00.000.000.4.1.5
Copyright (c) 2014-2022 Illumina, Inc.
Command Line: --bcl-input-directory miseq-gex --output-directory gex-fastqs --sample-sheet gex_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.

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

To follow along:

• Download the tiny-bcl-atac-2.0.0.tar.gz file
• Download the tiny-bcl-gex-2.0.0.tar.gz file
• Uncompress the tar files by running:

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

• Download the ATAC sample sheet
• Download the GEX sample sheet

GEX library

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bcl-convert --bcl-input-directory /working-directory/miseq-gex \
--output-directory /working-directory/gex-fastqs \
--sample-sheet /working-directory/cellranger-arc-tiny-bcl-gex-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.

A folder called gex-fastqs is created in the working directory. This folder contains your newly created FASTQ files.

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.
├── gex-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_gex_S1_L001_R1_001.fastq.gz
│   ├── test_sample_gex_S1_L001_R2_001.fastq.gz
│   ├── Undetermined_S0_L001_R1_001.fastq.gz
│   └── Undetermined_S0_L001_R2_001.fastq.gz
├── gex_samplesheet.csv
├── runinfo_gex.txt
└── runinfo.txt

ATAC library

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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.

A folder called atac-fastqs is created in the working directory. This folder contains your newly created FASTQ files.

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.
├── 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

Please contact Illumina Support at techsupport@illumina.com for general questions about BCL Convert. Or refer to BCL Convert documentation:

• BCL Convert User Guides
• BCL Convert Software Downloads

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