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Direct Demultiplexing with Illumina Software

Direct Demultiplexing with Illumina Software

This guide walks you through generating Cell Ranger-compatible FASTQs with BCL Convert.

Demultiplexing Chromium data with Illumina's BCL Convert software requires the correct specification of the sample sheet and command line options.

The Multiome GEX library is dual-indexed. This section describes how to configure 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, bcl-convert requires 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.

Illumina's BCL Convert is a software application that converts BCL files into FASTQ files. This page explains how to use BCL Convert for Chromium Epi 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.

Please contact Illumina Support for questions about BCL Convert versions, or troubleshooting help. Refer to BCL Convert documentation for download and installation steps:

  • BCL Convert User Guides
  • BCL Convert Software Downloads. Available as an RPM package. An Illumina account is required for download.

See the 10x Genomics Knowledge Base article for additional installation help.

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.

Do not trim adapters during demultiplexing. Leave these settings blank. Trimming adapters from reads can potentially damage 10x Barcodes and UMIs, resulting in pipeline failure or data loss.


If you are using an Illumina sample sheet for demultiplexing with bcl-convert, please remove these lines under the [Settings] section Adapter or AdapterRead1 or AdapterRead2.

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 nameDescription
    LaneOptional. 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_IDThe sample ID
    indexI7 index sequence
    index2I5 index sequence
    Sample_ProjectOptional. 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:

[Header]
FileFormatVersion,2

[BCLConvert_Settings]
CreateFastqForIndexReads,0
TrimUMI,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 is required to tell 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.

[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:

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:

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:

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

GEX library

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.

.
├── 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

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.

.
├── 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

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