Sequencing Requirements for Single Cell Multiome ATAC + Gene Expression

Specifications, Last Modified on September 9, 2020, Permalink

The Chromium™ Single Cell Multiome ATAC + Gene Expression Solution produces Illumina® sequencer-ready libraries. Sequencing requirements for the two types of sequencing libraries produced are listed below.

Single Cell Multiome Gene Expression libraries

Supported Sequencers

Recommended Sequencing: Minimum 20,000 read pairs/cell*

Dual Indexed Sequencing Run: Single Cell Multiome Gene Expression libraries are dual-indexed. This means both i5 and i7 reads are used for demultiplexing. We do not recommend sequencing 10x Single Cell Multiome Gene Expression dual index libraries with a single-index configuration.

Read Read 1 i7 Index i5 Index Read 2
Purpose Cell barcode & UMI Sample Index Sample Index Insert
Length** 28 10 10 90

*Adjust sequencing depth for the required performance or application. The Sequencing Saturation metric and curve in the Cell Ranger ARC run summary can be used to optimize sequencing depth for specific sample types.

**Shorter transcript reads may lead to reduced transcriptome alignment rates. Cell barcode, UMI and Sample index reads must not be shorter than indicated. Any read can be longer than recommended. Additional bases in Sample index reads must be trimmed using cellranger mkfastq or Illumina's bcl2fastq prior to further analysis. Additional bases in Cell barcode or UMI reads will automatically be ignored by Cell Ranger ARC.

Further information on sequencing specifications and expected data metrics can be found in the Sequencing Metrics & Base Composition of SC3' v3.1 Dual Index Libraries technical note.

Single Cell Multiome ATAC Libraries

A special consideration that needs to be taken into account when sequencing Single Cell Multiome ATAC libraries is the dual-indexing workflow of the Illumina® sequencer to be used. In short, sequencers can perform dual-indexing in Forward Strand (Workflow A) and Reverse Complement Strand (Workflow B) workflows which determines the direction in which the i5 index is read. Therefore, sequencing length requirements for the i5 read will change depending on the Illumina® sequencing workflow for each specific sequencer. For more information about the workflows, visit the Illumina® website and refer to the Indexed Sequencing Overview Guide (15057455 v07).

Supported Sequencers:

Sequencer Indexing Workflow Notes
Illumina® NovaSeq Forward or Reverse Complement Strand Software v1.6 and v1.0 kits run the Forward Strand workflow.
Software v1.7 and v1.5 run the Reverse Complement Strand workflow.
Follow recommendations for Reverse Complement Strand workflow.
Illumina® HiSeq 2500 Forward Strand Follow recommendations for Forward Strand workflow.
Illumina® NextSeq 500/550 Reverse Complement Strand Does not support longer i5 read.
Requires a custom recipe for sequencing the i5 index: 8 dark cycles, 16 cycles
Illumina® MiSeq Forward Strand Follow recommendations for Forward Strand workflow.

Recommended Sequencing Depth: 25,000 read pairs per nucleus (50,000 individual reads. 25,000 from R1, 25,000 from R2)*

Dual-Indexed Sequencing Run: Single Cell Multiome ATAC libraries are dual-indexed. Please note, only the i7 index read is used for demultiplexing. The i5 index read is used to capture the 10x barcode information.

PhiX Spike-In Recommendations: 1%.

Forward Strand (Workflow A) Sequencers:

Read Read 1 i7 Index i5 Index Read 2
Purpose Transposed DNA Sample Index 10x Barcode Transposed DNA
Length 50 ** 8 16 49 **

Reverse Strand (Workflow B) Sequencers:

Read Read 1 i7 Index i5 Index Read 2
Purpose Transposed DNA Sample Index 10x Barcode Transposed DNA
Length 50 ** 8 24 49 **

* Adjust sequencing depth for the required performance or application. The Sequencing Saturation metric and curve in the Cell Ranger ARC run summary can be used to optimize sequencing depth for specific sample types.

** Sequence length can can be adjusted based on sequencing kit used.

Further information on sequencing specifications and expected data metrics can be found in the Sequencing Metrics & Base Composition of Single Cell Multiome ATAC Libraries technical note.

For any additional help or clarification, contact Support at [email protected]