Sequencing Requirements for Single Cell V(D)J

Specifications, Last Modified on June 11, 2020, Permalink

The Chromium™ Single Cell V(D)J Solution produces up to four different Illumina® sequencer-ready libraries with unique sample indices:

Please note that the sample index plate required for Cell Surface Protein libraries is different from the sample index plate used for V(D)J Enriched and 5’ Gene Expression libraries.

For V(D)J Enriched and 5’ Gene Expression libraries, use ONLY Chromium i7 Sample Index Plate (PN-120262). For Cell Surface Protein library, use ONLY Chromium i7 Sample Index Plate N, Set A(PN-1000084). Consider sample index compatibility when pooling different libraries; unique sample index for each of the pooled libraries is required.

Our recommended sequencing read length configuration for pooling and sequencing all library types together is 26 x 91 bp, but other read lengths are also supported. Please see the individual specifications below for more information.

Supported Sequencers:

Single-Indexed Sequencing Run: Single Cell V(D)J Enriched, 5' Gene Expression and Cell Surface Protein libraries are single-indexed. We do not recommend sequencing these libraries with a dual-index configuration. **

PhiX Spike-In Recommendations: 1%

* Sequencing Single Cell V(D)J enriched, Feature Barcoding, or 5' Gene Expression libraries on the NextSeq 500/550 platform may yield reduced sequence quality and sensitivity relative to the MiSeq, HiSeq and NovaSeq platforms.

** 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.


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

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

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

*** 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 Enriched libraries (alone or in combination with the 5' Gene Expression and/or Cell Surface Protein libraries) may be sequenced at 150 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 5' Gene Expression Libraries (v1 and v1.1)

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

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 (This metric was named cDNA PCR Duplication in Cell Ranger 1.1 or earlier).

***** 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 Feature Barcoding Libraries (v1 and v1.1)

Minimum Sequencing Depth: 5,000 read pairs/cell

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

****** 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 V(D)J Cell Surface Protein libraries independently, they may also be sequenced in a 26 x 8 x 25 bp configuration.