10x Genomics Support/Space Ranger/Tutorials/

Demultiplexing with Illumina Software

Demultiplexing Visium data with Illumina's BCL Convert or bcl2fastq software requires the correct specification of the sample sheet and command line options. This page will walk you through generating Space Ranger-compatible FASTQs with BCL Convert and bcl2fastq.

Both bcl2fastq and BCL Convert are currently available. BCL Convert is a newer demultiplexing software. Click here for more information on Illumina's website.

This section describes how to configure bcl2fastq or BCL Convert for libraries created with the dual indexing kits. All spatial libraries use dual index sets.

These are unique dual-indexing sample indices. This means there is a unique sample index barcode in both the i7 and i5 index reads (also known as I1 and I2). 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.

Download the dual index kit sample index reference files at these links:

KitLibrary
Dual Index Kit TT, Set AVisium Spatial Gene Expression
Dual Index Kit TS, Set AVisium Spatial Gene Expression for FFPE
Dual Index Kit TS, Set AVisium CytAssist Spatial Gene Expression for FFPE
Dual Index Kit TS, Set AVisium CytAssist Spatial Gene Expression for Fresh Frozen

The index sequence in the sample index reference file should be entered into the index column of the bcl2fastq or BCL Convert 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 used (learn more here):

  • index2_workflow_a: NovaSeq™ 6000 v1, MiSeq™, MiniSeq™ with Rapid Reagent Kits, HiSeq™ 2500, and HiSeq™ 2000.
  • index2_workflow_b: NovaSeq™ 6000 v1.5, iSeq™ 100, MiniSeq™ with Standard Reagent Kits, NextSeq™, HiSeq™ X, and HiSeq™ 3000/4000. More information about dual indexing is available in the Illumina Indexed Sequencing Overview Guide.

The bcl2fastq software is available for download and installation on the Illumina Support if you have questions about bcl2fastq versions, or for help troubleshooting its download and installation.

You will need to create a sample sheet in order to get bcl2fastq to correctly embed the names of samples into output FASTQ files. 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-A1" for Fresh Frozen samples or "S1-TS-A1" for FFPE samples).

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

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 with bcl2fastq, BCL Convert or our mkfastq pipeline, please remove these lines under the [Settings] section: Adapter or AdapterRead1 or AdapterRead2.

Dual index sample sheet

For each sample, enter its lane, sample name, and sample index set into the Illumina bcl2fastq sample sheet. Here is an example using SI-TT-A1 indices for i7 and index2_workflow_b i5:

[Data] Lane,Sample_ID,index,index2 1,s1,GTAACATGCG,AGGTAACACT

Illumina bcl2fastq must be called with the correct --use-bases-mask argument, and other arguments, in order to properly demultiplex and output FASTQs for all the reads in a Visium library.

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.

For bcl2fastq2 v2.20, these are the most common command line formats for sequencers running RTA 1.18.54 and higher for dual index kits (edit the file paths for your data).

SAMPLE_SHEET_PATH=/path/to/sample/sheet/csv OUTPUT_DIR=/path/to/save/outputs FLOWCELL_DIR=/path/to/input/BCL/files INTEROP_DIR=./stats bcl2fastq --use-bases-mask=Y28,I10,I10,Y91 \ --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 nine bases, you will need to truncate the last base in order for Space Ranger 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:

ReadDesiredActualArgument
i7 Index Read (I1)89I8n

A new folder is created (name specified by the --output-dir flag). This folder contains the FASTQ file sets, statistics, and reports.

A convenient way to test bcl2fastq is by downloading the iseq-DI example dataset. This dual-indexed iSeq dataset has been selected for its small size (541 MB). The example below is applicable to Visium libraries, 3' Single Cell Gene Expression, 5' Immune Profiling, and Fixed RNA Profiling libraries processed with the TT Set A dual index kit. It should not be used to run downstream pipelines (e.g. spaceranger count).

To follow along,

tar -xvf /working-directory/iseq-DI.tar.gz

Run bcl2fastq

Run bcl2fastq (remember to customize the /working-directory/ path with the path to your input/output directory).

SAMPLE_SHEET_PATH=/working-directory/bcl2fastq_samplesheet.csv OUTPUT_DIR=/working-directory/tiny-FASTQs FLOWCELL_DIR=/working-directory/iseq-DI INTEROP_DIR=/working-directory/stats bcl2fastq --use-bases-mask=Y28,I10,I10,Y91 \ --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}

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

tiny-FASTQs/ ├── Reports │ └── html ├── Stats │ ├── AdapterTrimming.txt │ ├── ConversionStats.xml │ ├── DemultiplexingStats.xml │ ├── DemuxSummaryF1L1.txt │ ├── FastqSummaryF1L1.txt │ └── Stats.json ├── iseq-DI_S1_L001_I1_001.fastq.gz ├── iseq-DI_S1_L001_I2_001.fastq.gz ├── iseq-DI_S1_L001_R1_001.fastq.gz ├── iseq-DI_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

Illumina's BCL Convert is another software application that converts BCL files into FASTQ files. This page explains how to use BCL Convert for 10x Genomics products 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 Space Ranger.

BCL Convert is available for download and installation on the Illumina Support if you have questions about BCL Convert versions, or need help troubleshooting its download and installation.

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 with bcl2fastq, BCL Convert or our mkfastq pipeline, 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 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.
  • [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.

Dual index

This section shows an example sample sheet for libraries created with the Dual Index Plate TT, Set A or Dual Index Plate TS, Set A. The parameter CreateFastqForIndexReads,0 under [BCLConvert_Settings] tells BCL Convert not to generate FASTQ files for indices. Space Ranger does not require FASTQ files for indices.

[Header] FileFormatVersion,2 [BCLConvert_Settings] CreateFastqForIndexReads,0 [BCLConvert_Data] Lane,Sample_ID,index,index2 1,test_sample,GTAACATGCG,AGGTAACACT

The command to run bcl-convert:

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

The required arguments are:

  • --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.

This is the expected output after a successful BCL Convert run.

bcl-convert Version 00.000.000.3.10.5 Copyright (c) 2014-2018 Illumina, Inc. Command Line: --bcl-input-directory iseq-DI --output-directory tiny-FASTQs --sample-sheet bcl_convert_samplesheet.csv # CPU hw threads available: 48 Parallel Tiles: 1. Threads Per Tile: 48 SW compressors: 48 SW decompressors: 24 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 iseq-DI example dataset. This dual-indexed iSeq dataset has been selected for its small size (541 MB). The example below applies to Visium libraries, 3' Single Cell Gene Expression, 5' Immune Profiling, and Fixed RNA Profiling libraries processed with the TT Set A dual index kit. It should not be used to run downstream pipelines (e.g. spaceranger count). To follow along:

tar -xf /working-directory/iseq-DI.tar.gz

Run BCL Convert

bcl-convert --bcl-input-directory /working-directory/iseq-DI \ --output-directory /working-directory/tiny-FASTQs \ --sample-sheet /working-directory/bcl_convert_samplesheet.csv

Remember to customize the --bcl-input-directory path with the path to your input directory.

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

tiny-FASTQs/ ├── Logs │   ├── Errors.log │   ├── FastqComplete.txt │   ├── Info.log │   └── Warnings.log ├── Reports │   ├── Adapter_Metrics.csv │   ├── Demultiplex_Stats.csv │   ├── fastq_list.csv │   ├── Index_Hopping_Counts.csv │   ├── IndexMetricsOut.bin │   ├── Quality_Metrics.csv │   ├── RunInfo.xml │   ├── SampleSheet.csv │   └── Top_Unknown_Barcodes.csv ├── iseq-DI_S1_L001_R1_001.fastq.gz ├── iseq-DI_S1_L001_R2_001.fastq.gz ├── Undetermined_S0_L001_R1_001.fastq.gz └── Undetermined_S0_L001_R2_001.fastq.gz

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

After generating FASTQs, you are ready to run the spaceranger count pipeline following instructions on this page.