10x Genomics libraries contain P5 and P7 adapters, which can be used for Illumina sequencing. These libraries include 16 bp 10x GEM Barcodes (10x Barcode) encoded at the start of TruSeq Read 1 (Read 1T). Sample index sequences are incorporated as the i5 and i7 index reads. 10x Genomics assays can also be modified to enable sequencing on various long- and short-read sequencing platforms, with some platforms requiring third-party analysis tools.

Some variation in assay performance is expected based on the sequencer choice.

The 10x Genomics Compatible Products page contains a list of sequencing platforms compatible with 10x Genomics libraries.

The sequencers listed below have been verified as compatible with the indicated library type. Sequencer compatibility testing was performed either directly by 10x Genomics or by the Compatible Partner with data review by 10x Genomics.

Not all library types have been tested with every sequencer. For clarity, the tables below indicate which library types were tested and which have not been tested. Compatibility cannot be guaranteed with library types that were not tested.

The Sequencing Handbook CG000809 contains additional resources for various 10x Genomics libraries.

Illumina sequencers - short-read sequencing

* Recommended loading concentrations and % PhiX are provided based on internal testing. These recommendations are suggested starting points and may need adjustments. Loading concentration recommendations are based on qPCR quantification; alternative quantification methods may affect optimal loading concentrations. Higher PhiX is recommended for Multiplex libraries than Flex Singleplex libraries.

Library Sequencing Parameters

Sequencing configurations for Flex Gene Expression and Protein Expression are described below. For Flex experiments including CRISPR Guide Capture, refer to the respective Technical Note.

Read 1 Sequencing Configuration: This configuration reads the sequences required for sample demultiplexing in Read 1.

GEM-X Flex v2

*Flex v2 Sequencing Depth for High Cell/Nuclei Inputs

If an experiment targets more than 320,000 cells or nuclei, the Pre-Amplified material will be split into 4 separate SI-PCR reactions for library construction.

Sequencing Example

For example, for a Gene Expression experiment targeting 1,000,000 (1M) cells. The sequencing depth requirement (10,000 read pairs per cell) applies to the total pool of material representing the entire cell population. How users proceed depends on whether they pool the 4 separate libraries.

Total minimum recommended number of reads:

• 1M cells * 10,000 read pairs per cell = 10,000,000,000 (10B) total reads

If the 4 libraries are pooled together (preferred and recommended method):

• Sequence the library pool to a minimum depth of 10B reads
  • Each of the 4 libraries will receive ~ 2.5B reads and cumulative depth across all libraries will equate to the minimum recommended sequencing depth of 10,000 read pairs per cell

If the 4 libraries are not pooled together:

• The total read requirement is quartered and applied to each of the separate libraries:
  • 10B total reads / 4 total libraries = 2.5B reads per library
• This is equivalent to sequencing each of the 4 libraries at a depth of 2,500 read pairs per cell:
  • 1M cells * 2,500 read pairs per cell = 2.5B reads
• By sequencing each of the libraries at 2.5B reads, the cumulative depth across all libraries will equate to the minimum recommended sequencing depth of 10,000 read pairs per cell

When sequencing GEM-X Flex v2 Gene Expression libraries on a NovaSeq 6000 instrument (Standard workflow) with a loading concentration of 150 pM, a comparison of Read 1 (Read 1: 54 cycles, i7 index: 10 cycles, i5 index: 10 cycles, Read 2: 50 cycles) and Read 2 (Read 1: 28 cycles, i7 index: 10 cycles, i5 index: 10 cycles, Read 2: 90 cycles) sequencing configurations indicates that their performance is similar.

GEM-X Flex v1

When sequencing GEM-X Flex v1 Gene Expression libraries on a NovaSeq 6000 instrument (Standard workflow) with a loading concentration of 150 pM, a comparison of Read 1 (Read 1: 48 cycles, i7 index: 10 cycles, i5 index: 10 cycles, Read 2: 50 cycles) and Read 2 (Read 1: 28 cycles, i7 index: 10 cycles, i5 index: 10 cycles, Read 2: 90 cycles) sequencing configuration indicates that their performance is similar.

Read 2 Sequencing Configuration: This configuration reads the sequences required for sample demultiplexing in Read 2. Note that this read configuration is not compatible in the following special cases where the sequences required for sample demultiplexing do not occur in the same positions when read in Read 2: multiplexing experiment Protein Expression libraries derived from using Proteintech Group (PTG) antibodies mixed with Biolegend antibodies (see this Q&A for details); multiplexing experiment using Custom Probes designed for Flex v1 that omit the Constant Sequence-NN. For these special cases, sequence with the Read 1 Sequencing Configuration.

GEM-X Flex v2

*Flex v2 Sequencing Depth for High Cell/Nuclei Inputs

If an experiment targets more than 320,000 cells or nuclei, the Pre-Amplified material will be split into 4 separate SI-PCR reactions for library construction.

Sequencing Example

For example, for a Gene Expression experiment targeting 1,000,000 (1M) cells. The sequencing depth requirement (10,000 read pairs per cell) applies to the total pool of material representing the entire cell population. How users proceed depends on whether they pool the 4 separate libraries.

Total minimum recommended number of reads:

• 1M cells * 10,000 read pairs per cell = 10,000,000,000 (10B) total reads

If the 4 libraries are pooled together (preferred and recommended method):

• Sequence the single pooled library to a minimum depth of 10B reads
  • Each of the 4 libraries will receive ~ 2.5B reads and cumulative depth across all libraries will equate to the minimum recommended sequencing depth of 10,000 read pairs per cell

If the 4 libraries are not pooled together:

• The total read requirement is quartered and applied to each of the separate libraries:
  • 10B total reads / 4 total libraries = 2.5B reads per library
• This is equivalent to sequencing each of the 4 libraries at a depth of 2,500 read pairs per cell:
  • 1M cells * 2,500 read pairs per cell = 2.5B reads
• By sequencing each of the libraries at 2.5B reads, the cumulative depth across all libraries will equate to the minimum recommended sequencing depth of 10,000 read pairs per cell

†Minimum required Read 2 length is 89 bp

GEM-X Flex v1

*Minimum required Read 2 length is 76 bp

The two schemes for GEM-X Flex v1 and v2 libraries can be used interchangeably. However, for effective data analysis, it may be important to understand the specific conditions under which Cell Ranger extracts information from Read 1 versus Read 2, as well as the rationale behind these selections. See the Technical Note Sequencing Metrics & Base Composition of Chromium Flex Libraries (CG000677) for more information.

Library Pooling

Flex Gene Expression and Protein Expression libraries may be pooled for sequencing, taking into account the differences in cell number and per-cell read depth requirements between each library. Samples utilizing the same sample index should not be pooled together or run on the same flow cell lane, as this would not enable correct sample demultiplexing.

Ultima Genomics sequencers - short-read sequencing

Element Biosciences sequencers - short-read sequencing