.

Related articles

Which 10x Genomics assays are compatible with long-read sequencing applications?

Question: Which 10x Genomics assays are compatible with long-read sequencing applications?

Answer: Amplified cDNA from 10x Genomics assays can be adapted for sequencing on long-read sequencing applications. This article provides information related to testing of 10x Genomics assays with long-read sequencing applications. 

Background

Combining long-read sequencing with single cell or spatial assays enables the unambiguous identification of alternative splicing at single cell or spatial resolution. Unique isoforms associated with specific cell types can be distinguished which are otherwise undetectable using short-read sequencing. Long-read sequencing may also offer advantages in variant detection, including structural variants. 

Adapting cDNA from 10x Genomics assays for sequencing on long-read platforms requires third party reagents and third party protocols. Contact the relevant Compatible Partner for guidance and troubleshooting related to protocol modifications after cDNA amplification. Also contact the relevant Compatible Partner for guidance and troubleshooting related to sequencing configurations, sequencing primers, library pooling, and sequencing performance.

10x Genomics assays have not been optimized for long-read sequencing; thus, 10x Genomics cannot guarantee that sufficient cDNA will be generated to use as input into library preparation for long-read sequencing. 10x Genomics also cannot guarantee that sufficient full-length transcripts will be captured. cDNAs generated using 10x assays are expected to contain some full-length transcripts as well as some non-full length transcripts due to sample quality, potential internal priming, and other factors. Refer to the 10x Genomics Technical Note on Interpreting Intronic and Antisense Reads in 10x Genomics Single Cell Gene Expression Data for additional information. 10x Genomics will not offer replacement reagents related to insufficient cDNA yield for long-read library preparation or insufficient full-length transcripts in the final data. 

10x Genomics does not currently offer tools for analyzing long-read sequencing data. Contact the relevant Compatible Partner for questions related to third party tools for analysis of long-read sequence data.

Oxford Nanopore Technologies

10x Genomics cDNA can be adapted for sequencing on Oxford Nanopore Technologies (ONT) PromethION™ Sequencing Devices. Full length barcoded and amplified cDNA from 10x Genomics assays is biotin-labeled and enriched via a streptavidin-biotin pulldown to deplete PCR artifacts. The cDNA is then further amplified and adapters are ligated to generate long-read sequencing libraries.

ONT Ligation Sequencing Kit V14 - SQK-LSK114

The Ligation Sequencing Kit V14 (SQK-LSK114), PCR Expansion Kit (EXP-PCA001) and custom-ordered oligos can be used to adapt 10x Genomics cDNA for Oxford Nanopore sequencing, as described in the following ONT protocols for 3’ cDNA and 5’ cDNA. These protocols require 10 ng of high quality cDNA per sample.  

This approach was tested as part of the 10x Genomics Compatible Partner Program and verified as compatible with cDNA from the following 10x Genomics assays:

  • Universal 3’ Gene Expression - GEM-X 3’ v4 and Next GEM 3’ v3.1
  • Universal 5’ Gene Expression - GEM-X 5’ v3 and Next GEM 5’ v2

Datasets for GEM-X 3’ v4 and 5’ v3 are available here: https://epi2me.nanoporetech.com/sc-gemx-2025.02/

Notes regarding other 10x Genomics assays:

  • It may be possible to use the ONT 3’ cDNA protocol with cDNA from the 10x Genomics assays below that share the same cDNA PCR handles as the Universal 3’ Gene Expression assay. However, this has not been tested nor validated by 10x Genomics.
    • Visium Spatial Gene Expression for Fresh Frozen assay (v1)
    • Epi Multiome ATAC + Gene Expression assay (cDNA component only, not ATAC)

PCR-cDNA Sequencing Kit - SQK-PCS111 [legacy product]

Prior testing through the 10x Genomics Compatible Partner Program was performed on the legacy PCR-cDNA Sequencing Kit - SQK-PCS111. Datasets and Application Notes related to this previous kit version are included below for reference. 

Data analysis

Analysis requires 3rd party tools. ONT has developed a tool called Workflow Single Cell (wf-single-cell). Further information is available at https://labs.epi2me.io/workflows/wf-single-cell/ and https://github.com/epi2me-labs/wf-single-cell.

Contact ONT (support@nanoporetech.com) for assistance with troubleshooting ONT protocols and sequencing, and for questions related to data analysis.

Pacific Biosciences

10x Genomics cDNA can be adapted for sequencing on Pacific Biosciences (PacBio) long-read sequencing systems. Full length barcoded and amplified cDNA from 10x Genomics assays is concatenated into larger fragment libraries for long-read sequencing. HiFi reads generated from sequencing the concatenated molecules can then be used to bioinformatically infer the original molecules.

Kinnex method for adapting 10x Genomics cDNA

  • The Kinnex single-cell RNA kit was designed for use with PacBio’s Sequel II, Sequel IIe, Vega and Revio sequencing systems, and has been tested by PacBio with cDNA from the 10x Genomics assays listed below. This approach requires 15 to 75 ng of high quality amplified 10x Genomics cDNA as input. This approach has not been validated by 10x Genomics.
    • Universal 3’ Gene Expression - Next GEM 3’ v3.1
    • Universal 5’ Gene Expression - Next GEM 5’ v2

MAS-Seq method for adapting 10x Genomics cDNA [discontinued]

cDNA from other 10x Genomics assays

GEM-X Universal 3’ and 5’

  • The testing mentioned above was performed on Next GEM assay versions. It may be possible to use Kinnex kits with cDNA from GEM-X Universal 3’ v4 and 5’ v3 assays as the cDNA shares the same PCR handles as 3’ v3.1 and 5’ v2 assays. This has not been validated by 10x Genomics. Minor changes would be required during data analysis, to account for updated 10x Genomics barcodes in the new GEM-X assay version. Refer to the PacBio SMRT Link Kinnex single-cell troubleshooting guide for further details. 

Visium Spatial Gene Expression for Fresh Frozen (v1) 

  • It may be possible to use Kinnex kits with cDNA from the 10x Genomics Spatial Gene Expression for Fresh Frozen (v1) assay as it shares the same cDNA PCR handles as the Universal 3’ assay. This has not been validated by 10x Genomics. Analysis would require advanced parameter changes in the SMRT Link analysis pipeline and is not supported by PacBio. Refer to the PacBio SMRT Link Kinnex single-cell troubleshooting guide for further details. 

Epi Multiome ATAC + Gene Expression assay

  • It may be possible to use Kinnex kits with cDNA from the Epi Multiome ATAC + Gene Expression assay (cDNA component only, not ATAC) as the cDNA shares the same cDNA PCR handles as the Universal 3’ assay. This has not been validated by 10x Genomics or PacBio.

Data analysis

Analysis requires 3rd party tools. PacBio provides a Read Segmentation and Single-cell Iso-Seq workflow as part of their SMRT Link software. Refer to the PacBio SMRT Link Kinnex single-cell troubleshooting guide.

Contact PacBio (support@pacb.com) for assistance with troubleshooting PacBio protocols and sequencing, and for questions related to data analysis. 

Considerations for V(D)J, Feature Barcode, and probe-based assays

The ONT and PacBio testing mentioned in this article was performed on full length cDNA. 10x Genomics has not performed testing on other library types. 

10x Genomics probe-based libraries (e.g. Flex Gene Expression) and Feature Barcode libraries (e.g. Cell Surface Protein) do not contain full length transcripts and therefore may be less suitable for long-read sequencing applications. Furthermore, these libraries contain different PCR handles that would not be recognized by the primers in the ONT and PacBio conversion kits mentioned in this article. 

For the Universal 5’ Gene Expression assay, enriched cDNA can be generated for V(D)J analysis. V(D)J-enriched cDNA has different PCR handles and would not be recognized by the primers in the ONT and PacBio conversion kits mentioned in this article. Alternative approaches would be required if long read sequencing of V(D)J-enriched fragments is desired. As one example, see Singh et al 2019 which uses a target capture approach for V(D)J enrichment, followed by ONT 1D Adapter Ligation Sequencing Kit and long-read sequencing, to obtain enriched full length V(D)J sequences from the 10x Genomics Universal 3' assay.

Publications for long-read sequencing

There are several customer publications demonstrating the use of long-read sequencing with 10x Genomics assays. A few of these publications are highlighted below:

Single Cell Gene Expression 

Immune profiling

Spatial Gene Expression

 

Products: Universal 3' Gene Expression, Universal 5' Gene Expression, Visium for Fresh Frozen, Epi Multiome ATAC + Gene Expression