Sequencing Requirements for Immune Profiling HT

Specifications, Last Modified on June 23, 2022, Permalink

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

Supported Sequencers:

PhiX Spike-In Recommendations: 1%

Single Cell 5' HT v2 Dual Index Gene Expression Libraries

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

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

Single Cell 5' HT v2 Dual Index Cell Surface Protein Libraries

Minimum Sequencing Depth: 5,000 read pairs/cell

Dual-Indexed Sequencing Run: Single Cell 5' HT v2 Dual Index Cell Surface Protein are dual-indexed. We do not recommend sequencing 10x Single Cell 5' HT 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' HT 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.

The Single Cell 5' HT v2 Dual Index application is not supported with Cell Multiplexing libraries.