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

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 software 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.

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 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 the 10x Barcodes and the UMIs, resulting in pipeline failure or data loss. If you are using an Illumina sample sheet for demultiplexing, please remove these lines under the [Settings] section: Adapter, 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.

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.

[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. 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-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.

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

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