Sequencing Requirements for Single Cell V(D)J

Specifications, Last Modified on June 14, 2022, Permalink

The Chromium™ Single Cell Immune Profiling Solution produces up to four different Illumina® sequencer-ready libraries:

Supported Sequencers:

PhiX Spike-In Recommendations: 1%

Single Cell 5' v2 Dual Index Gene Expression Libraries

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

Dual-Indexed Sequencing Run: Single Cell 5' v2 Dual Index libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 5’ v2 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 10x™ Barcode, UMI Sample Index Sample Index Insert
Length** 26 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 5' v2 Dual Index V(D)J Libraries

Minimum Sequencing Depth: 5,000 read pairs/targeted cell (for more information please refer to this guide).

Dual-Indexed Sequencing Run: Single Cell 5' v2 Dual Index V(D)J libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 5' v2 Dual Index V(D)J libraries with a single-index configuration.

Read Read 1 i7 Index i5 Index Read 2
Purpose 10x™ Barcode, UMI Sample Index Sample Index Insert
Length* 26 10 10 90

* Shorter reads than indicated above can lead to decreased application performance. In particular, Read 2 length is critical for spanning the V-J junctions. 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. Please see the following article for more options for sequencing 5' v2 Dual Index V(D)J libraries: Can I sequence my 5' v2 Dual Index V(D)J libraries in a different sequencing configuration than the ones recommended in the user guide?

Single Cell 5' v2 Dual Index Cell Surface Protein Libraries

Minimum Sequencing Depth: 5,000 read pairs/cell

Dual-Indexed Sequencing Run: Single Cell 5' v2 Dual Index Cell Surface Protein are dual-indexed. We do not recommend sequencing 10x Single Cell 5' v2 Dual Index Cell Surface Protein libraries with a single-index configuration.

Read Read 1 i7 Index i5 Index Read 2
Purpose 10x™ Barcode, UMI Sample Index N/A Insert
Length* 26 10 10 90

* 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.

If sequencing Single Cell 5' v2 Dual Index Cell Surface Protein libraries independently, they may also be sequenced in a 26 x 10 x 10 x 25 bp configuration.

Single Cell 5' v2 Dual Index CRISPR Screening Libraries

Minimum Sequencing Depth: 5,000 read pairs/cell

Dual-Indexed Sequencing Run: Single Cell 5' v2 Dual Index CRISPR Screening libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 5' v2 Dual Index CRISPR Screening libraries with a single-index configuration. We also do not recommend sequencing 10x Single Cell 5' v2 Dual Index CRISPR Screening libraries alone; 5' CRISPR Screening libraries should be pooled with Gene Expression libraries to increase base diversity.

Read Read 1 i7 Index i5 Index Read 2
Purpose 10x™ Barcode, UMI Sample Index N/A Insert
Length* 26 10 10 90

* 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.

Single Cell 5' Gene Expression Libraries (v1 and v1.1)

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

Single-Indexed Sequencing Run: Single Cell 5' Gene Expression v1 and v1.1 libraries are single-indexed. We do not recommend sequencing these libraries with a dual-index configuration.**

Read Read 1 i7 Index i5 Index Read 2
Purpose 10x™ Barcode, UMI Sample Index N/A Insert
Length *** 26 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.

** If a dual-index configuration is used, please use bcl2fastq's --use-bases-mask or mkfastq's --ignore-dual-index option to ignore the I2 read.

*** Shorter transcript reads may lead to reduced transcriptome alignment rates. 10x™ 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.

Single Cell V(D)J Libraries (v1 and v1.1)

Minimum Sequencing Depth: 5,000 read pairs/targeted cell (for more information please refer to this guide).

Single-Indexed Sequencing Run: Single Cell V(D)J v1 and v1.1 libraries are single-indexed. We do not recommend sequencing these libraries with a dual-index configuration.*

Read Read 1 i7 Index i5 Index Read 2
Purpose 10x™ Barcode, UMI Sample Index N/A Insert
Length** 26 8 0 91

* If a dual-index configuration is used, please use bcl2fastq's --use-bases-mask or mkfastq's --ignore-dual-index option to ignore the I2 read.

** Shorter reads than indicated above can lead to decreased application performance. In particular, Read 2 length is critical for spanning the V-J junctions. 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.

V(D)J v1/v1.1 libraries (alone or in combination with the 5' Gene Expression v1/v1.1 and/or Cell Surface Protein v1/v1.1 libraries) may be sequenced at 150 x 8 x 0 x 150 bp. At this read length configuration, our recommended minimum is 2,000 read pairs/targeted cell. Please see our page on Experiment design for V(D)J libraries for more information on sequencing recommendations.

Single Cell V(D)J Cell Surface Protein (v1 and v1.1)

Minimum Sequencing Depth: 5,000 read pairs/cell

Single-Indexed Sequencing Run: Single Cell V(D)J Cell Surface Protein v1 and v1.1 libraries are single-indexed. We do not recommend sequencing these libraries with a dual-index configuration.*

Read Read 1 i7 Index i5 Index Read 2
Purpose 10x™ Barcode, UMI Sample Index N/A Insert
Length** 26 8 0 91

* If a dual-index configuration is used, please use bcl2fastq's --use-bases-mask or mkfastq's --ignore-dual-index option to ignore the I2 read.

** 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.

If sequencing Single Cell V(D)J Cell Surface Protein v1 or v1.1 libraries independently, they may also be sequenced in a 26 x 8 x 0 x 25 bp configuration.