10x Genomics Support/Cell Ranger/Tutorials/

Generating FASTQs with BCL Convert (Illumina Software)

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 Cell Ranger.

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 [email protected] for 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 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.

  • [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 libraries created with the Dual Index Plate TT, Set A or Dual Index Plate TN, 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.

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

This section shows an example sample sheet for libraries created with the Single Index Kit T Set A and Single Index Kit N Set A. Other parameters are the same as the dual index example above. Since lane is not specified in this example, indices are searched in all lanes.

[Header] FileFormatVersion,2 [BCLConvert_Settings] CreateFastqForIndexReads,0 [BCLConvert_Data] Sample_ID,index Sample2_SI,GGTTTACT Sample2_SI,CTAAACGG Sample2_SI,TCGGCGTC Sample2_SI,AACCGTAA

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>

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:

bcl-convert Version 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 3' Single Cell Gene Expression, 5' Immune Profiling, Fixed RNA Profiling, and Visium libraries processed with the TT Set A dual index kit. It should not be used to run downstream pipelines (e.g. cellranger 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

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