Sequencing Requirements for Single Cell 3'

Specifications, Last Modified on July 7, 2020, Permalink

Single Cell 3' v3.1 Dual Index Gene Expression

The Chromium™ Single Cell Gene Expression Solution with Feature Barcoding technology produces Illumina® sequencer-ready libraries.

Supported Sequencers

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

Dual Indexed Sequencing Run: Single Cell 3' v3.1 Dual Index libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 3’ v3.1 Dual Index libraries with a single-index configuration. See: Can single index and dual index libraries be pooled for sequencing?

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 run summary can be used to optimize sequencing depth for specific sample types (note: this metric was named cDNA PCR Duplication in Cell Ranger 1.1 or earlier).

**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 1.3 or later.

Single Cell 3' v3.1 Dual Index Gene Expression with Feature Barcoding technology

Supported Sequencers

Recommended Sequencing: Minimum 5,000 read pairs/cell*

Dual Indexed Sequencing Run: Single Cell 3' v3.1 Dual Index Feature Barcode libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 3’ v3.1 Dual Index Feature Barcode libraries with a single-index configuration.

We recommend pooling Single Cell 3' v3.1 Dual Index Feature Barcode libraries with Single Cell 3' v3.1 Dual Index Gene Expression libraries to maintain nucleotide diversity during sequencing.

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 run summary can be used to optimize sequencing depth for specific sample types (note: this metric was named cDNA PCR Duplication in Cell Ranger 1.1 or earlier).

**If sequencing 3' v3.1 Dual Index Cell Surface Protein libraries independently, they may also be sequenced in a 28 x 10 x 10 x 25 bp configuration. We do not recommend sequencing Single Cell 3’ v3.1 Dual Index CRISPR Screening Libraries independently.

Single Cell 3' v3/v3.1 Gene Expression

The Chromium™ Single Cell Gene Expression Solution with Feature Barcoding technology produces Illumina® sequencer-ready libraries.

Supported Sequencers

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

Dual Indexed Sequencing Run: Single Cell 3' v3/v3.1 libraries are single-indexed. We do not recommend sequencing 10x Single Cell 3’ v3/v3.1 libraries with a dual-index configuration.

Read Read 1 i7 Index i5 Index Read 2
Purpose Cell barcode & UMI Sample Index N/A Insert
Length** 28 8 0 91

*Adjust sequencing depth for the required performance or application. The Sequencing Saturation metric and curve in the Cell Ranger run summary can be used to optimize sequencing depth for specific sample types (note: this metric was named cDNA PCR Duplication in Cell Ranger 1.1 or earlier).

**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 1.3 or later.


Single Cell 3' v3/v3.1 Gene Expression with Feature Barcoding technology

Supported Sequencers

Recommended Sequencing: Minimum 5,000 read pairs/cell*

Dual Indexed Sequencing Run: Single Cell 3' v3/v3.1 Feature Barcode libraries are single-indexed. We do not recommend sequencing 10x Single Cell 3’ v3/v3.1 Feature Barcode libraries with a dual-index configuration. We recommend pooling Single Cell 3' v3/v3.1 Feature Barcode libraries with Single Cell 3' v3/v3.1 Gene Expression libraries to maintain nucleotide diversity during sequencing.

Read Read 1 i7 Index i5 Index Read 2
Purpose Cell barcode & UMI Sample Index N/A Insert
Length** 28 8 0 91

*Adjust sequencing depth for the required performance or application. The Sequencing Saturation metric and curve in the Cell Ranger run summary can be used to optimize sequencing depth for specific sample types (note: this metric was named cDNA PCR Duplication in Cell Ranger 1.1 or earlier).

**If sequencing 3' v3/v3.1 Cell Surface Protein libraries independently, they may also be sequenced in a 28 x 8 x 25 bp configuration. If sequencing Single Cell 3’ v3/v3.1 CRISPR Screening Libraries independently, they may be sequenced in a 28 x 8 x 70 bp configuration.


Single Cell 3' v2

Supported Sequencers

Recommended Sequencing: 50,000 read pairs/cell*

Dual Indexed Sequencing Run: Single Cell 3' v2 libraries are single-indexed. We do not recommend to sequence 10x Single Cell 3’ v2 libraries with a dual-index configuration.

Read Read 1 i7 Index i5 Index Read 2
Purpose Cell barcode & UMI Sample Index N/A Insert
Length** 26 8 0 98

**If Single Cell 3' v2 libraries are sequenced with alternative sequencing formats, please refer to the Technical Note Base Composition of Sequencing Reads of Chromium™ Single Cell 3’ v2 Libraries for more details.