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Generating FASTQs with BCL Convert and bcl2fastq (Illumina Software)

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

  • Cell Ranger mkfastq is a 10x Genomics wrapper for bcl2fastq - Illumina's bcl2fastq - Illumina's BCL Convert Illumina's software may provide greater control over demultiplexing parameters.
Both bcl2fastq and BCL Convert are currently available. BCL Convert is a newer demultiplexing software. For more information, please check Illumina's website.

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 will walk you through generating Cell Ranger-compatible FASTQs with BCL Convert and bcl2fastq.

This section describes how to configure bcl2fastq or bcl-convert for libraries created with single and dual indexing kits.

This section describes how to configure for libraries created with the dual indexing kits.

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:

Dual Index Kit TT, Set A3' Gene Expression 5',

Gene Expression/VDJ/CRISPR Guide Capture, Fixed RNA Profiling Antibody Capture | | Dual Index Kit NT, Set A | 3' Antibody/CRISPR Guide Capture | | Dual Index Kit TN, Set A | 5' Antibody Capture | | Dual Index Kit NN, Set A | 3' Cell Multiplexing | | Dual Index Kit TS, Set A | Fixed RNA Profiling Gene Expression |

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 (more information 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.

This section describes how to configure bcl2fastq and bcl-convert for libraries created with single indexing kits. Download the single index kit sample index reference files at these links:

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 actually 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 bcl2fastq

Please contact Illumina Support at [email protected] for general questions about BCL Convert. Or refer to BCL Convert documentation:

After generating FASTQs, you are ready to run the cellranger count, cellranger vdj, or cellranger multi pipelines, depending on your data type and experimental design. Visit the Choosing a Pipelines page to find the Cell Ranger pipeline relevant to your data.