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

• V(D)J Enriched library (TCR and/or Ig)
• 5’ Gene Expression library
• Cell Surface Protein library
• 5' CRISPR Screening library
• Barcode Enabled Antigen Mapping (BEAM)*

*Feature Barcode technology for BEAM is not supported with the Single Cell Immune Cell Profiling 5' v3 assay.

Supported Sequencers:

• Illumina® NovaSeq 6000
• Illumina® HiSeq 3000/4000
• Illumina® HiSeq 2500 Rapid Run
• Illumina® NextSeq 500/550
• Illumina® NextSeq 1000/2000
• Illumina® MiSeq

PhiX Spike-In Recommendations: 1%

Single Cell 5' v3 Gene Expression Libraries

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

Dual-Indexed Sequencing Run: Single Cell 5' v3 libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 5’ v3 libraries with a single-index configuration. See: Can single index and dual index libraries be pooled for sequencing?

*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' v3 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' v3 V(D)J libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 5' v3 V(D)J libraries with a single-index configuration.

*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.

Single Cell 5' v3 Cell Surface Protein Libraries

Minimum Sequencing Depth: 5,000 read pairs/cell

Dual-Indexed Sequencing Run: Single Cell 5' v3 Cell Surface Protein are dual-indexed. We do not recommend sequencing 10x Single Cell 5' v3 Cell Surface Protein libraries with a single-index configuration.

*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' v3 Cell Surface Protein libraries independently, they may also be sequenced in a 28 x 10 x 10 x 25 bp configuration.

Single Cell 5' v3 CRISPR Screening Libraries

Minimum Sequencing Depth: 5,000 read pairs/cell

Dual-Indexed Sequencing Run: Single Cell 5' v3 CRISPR Screening libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 5' v3 CRISPR Screening libraries with a single-index configuration. We also do not recommend sequencing 10x Single Cell 5' v3 CRISPR Screening libraries alone; 5' CRISPR Screening libraries should be pooled with Gene Expression libraries to increase base diversity.

*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' 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?

*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.

* 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.

* 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.

* 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' v2 Dual Index Barcode Enabled Antigen Mapping (BEAM) Libraries

Minimum Sequencing Depth: 5,000 read pairs/cell

Dual-Indexed Sequencing Run: Single Cell 5' v2 Dual Index BEAM libraries are dual-indexed. We do not recommend sequencing 10x Single Cell 5' v2 Dual Index BEAM libraries with a single-index configuration. We also do not recommend sequencing 10x Single Cell 5' v2 Dual Index BEAM libraries alone; BEAM libraries should be pooled with Gene Expression libraries and/or V(D)J libraries to increase base diversity.

* 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. The minimum Read 2 length for 5’v2 BEAM libraries is 15 bp.

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.**

* 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.*

* 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.*

* 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.