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

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

Answer: The 10x Assays compatible with long-read sequencing applications are summarized below.

 

Summary of long-read sequencing compatibility with 10x Assays:

*Kinnex compatibility has not been verified by 10x Genomics. Kinnex compatibility has been tested by PacBio for 5’v2 and 3’v3.1. All other listed compatibilities have been verified by 10x Genomics except as listed. Please reach out directly to PacBio or ONT for additional information regarding compatibility of 10x assays with long-read sequencing.

 

The workflow modifications listed below will NOT work with ATAC, 5' V(D)J, Fixed RNA Profiling, Visium for FFPE, Visium CytAssist Spatial Gene Expression, or Visium HD Spatial Gene Expression workflows. 

 

Combining long-read sequencing with single cell assays enables the unambiguous identification of alternative splicing at single cell resolution.  Unique isoforms associated with specific cell types can be distinguished which are otherwise undetectable using short-read single-cell RNA-Seq. cDNAs generated using 10x assays will contain full-length transcripts as well as some non-full length transcripts due to sample quality, potential internal priming, and other factors. Please see our Interpreting Intronic and Antisense Reads in 10x Genomics Single Cell Gene Expression Data Technical Note for additional  information. While these workflows are customer enabled using workflows from10x Compatible Partners Oxford Nanopore Technologies (ONT) or Pacific Biosciences (PacBio), they are unsupported by 10x Genomics since they require workflow modifications and additional 3rd party reagents.

 

Please note: 10x Genomics will not be able to provide troubleshooting guidance for library generation, sequencing, or analysis using the PacBio or ONT workflows.  In addition, 10x Genomics cannot guarantee success with these unsupported downstream workflows.  We are unable to offer any replacements or goodwill reagents due to unsatisfactory results from PacBio or ONT workflows. This includes not having enough cDNA for use with these workflows.

 

PacBio:

Single-cell RNA sequencing using PacBio HiFi reads with the MAS-Seq method offers full-length isoform information at the single-cell level.  The MAS-Seq method uses a concatenation method for increasing throughput by joining cDNA molecules generated from single-cell platforms into longer concatenated fragments. HiFi reads generated from sequencing the concatenated molecules can then be used to bioinformatically inference the original molecules. The Kinnex kit uses the MAS-Seq method and adds compatibility for cDNA from 10x Genomics' 5' assay and for multiplexing up to four 10x libraries per run. Analysis requires 3rd party tools and is not supported by 10x Genomics. 

Materials required:

• Procedure & Checklist - Preparing MAS-Seq libraries using MAS-Seq for 10x Single Cell 3' kit.    
  • 15 to 75 ng of cDNA per sample
• Procedure & checklist - Preparing Kinnex libraries using Kinnex single-cell RNA kit                  
  • 15 to 75 ng of cDNA per sample

 

10x Assays compatible with MAS-Seq for 10x Single Cell 3’ kit:

• Chromium Single Cell Gene Expression (i.e. 3’ v3.1).
• Chromium Single Cell Immune Profiling (i.e. 5’ v2) and Spatial Gene Expression for Fresh Frozen (Visium for Fresh Frozen) assays are compatible, since the cDNA amplicons share the same PCR handles.  However, this requires advanced parameter changes in the SMRT Link analysis pipeline.  This has not been tested for performance by PacBio and is currently unsupported by PacBio.
  • Chromium Single Cell Immune Profiling (i.e. 5’ v2) assays require additional oligos for the TSO depletion step not currently provided in the MAS-Seq for 10x Single Cell 3’ kit that are described in the original method publication (Al’Khafaji et al.). Advanced parameter changes in the SMRT Link analysis pipeline are required. Performance of the 5’ v2 assays with the kit has not been by PacBio and is currently unsupported.

10x Assays compatible with Kinnex single-cell RNA kit:

• Chromium Single Cell Gene Expression (i.e. 3’ v3.1).
• Chromium Single Cell Immune Profiling (i.e. 5’ v2).
• Spatial Gene Expression for Fresh Frozen (Visium for Fresh Frozen) assay is compatible, since the cDNA amplicons share the same PCR handles.  However, this requires advanced parameter changes in the SMRT Link analysis pipeline.  This has not been tested for performance by PacBio and is currently unsupported by PacBio.

Additional Resources:

• MAS-Seq for 10x Single Cell 3’ Application Note: MAS-Seq for single-cell isoform sequencing
• Kinnex Application Note: Kinnex single-cell RNA kit for single cell isoform sequencing
• Kinnex Product page
• Analysis: PacBio provides a Read Segmentation and Single-cell Iso-Seq workflow as part of their SMRT Link software. 
• Press Releases: MAS-Seq for 3' press release and Kinnex for single cell press release
• For additional information, please reach out to PacBio (support@pacb.com).

 

 

Oxford Nanopore Technologies (ONT):

Long-read single-cell RNA sequencing is enabled using ONT PromethION™ Sequencing Devices. In this workflow, full-length cDNA is biotinylated and PCR amplified prior to library construction using the Oxford Nanopore cDNA-PCR Sequencing Kit.  Nanopore sequencing enables sequencing of full-length transcripts and splice variants. Analysis requires 3rd party tools and is not supported by 10x Genomics.

 

Materials required:

• Protocols, including materials and consumables: Single-cell transcriptomics with cDNA prepared using 10X Genomics
• PCR-cDNA Sequencing Kit (SQK-PCS111)
• 10 ng of cDNA required per sample [Please note: High quality cDNA is required for this workflow.  Highly fragmented RNA may affect library construction and assay performance.]
• Custom-ordered oligos [Details provided in Protocol linked above]

 

10x Assays compatible with ONT PromethION™ Sequencing Devices:

• Chromium Single Cell Gene Expression (i.e. 3’ v3.1).
• Chromium Single Cell Immune Profiling (i.e. 5’ v2) 
• Spatial Gene Expression for Fresh Frozen (Visium for Fresh Frozen)
• 10x Genomics Datasets:
• Chromium 3’ Single Cell Gene Expression
• Chromium 5’ Single Cell Gene Expression
• Visium Spatial Gene Expression

 

Additional Resources:

• 10x Genomics Application Note: Alternative transcript isoform detection with single cell and spatial resolution
• Analysis: Oxford Nanopore Technologies has developed a tool called Workflow Single Cell (wf-single-cell), formerly Sockeye. Available at: https://github.com/epi2me-labs/wf-single-cell.
• ONT Webinar: Single-cell transcriptomes with Oxford Nanopore
• ONT Note: Workflow: Single-cell transcriptomes
• Press Release: ONT announcement
• For additional information, please reach out to ONT (support@nanoporetech.com).

 

Publications:

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 

• High throughput error corrected Nanopore single cell transcriptome sequencing (ONT)
• Single-cell isoform RNA sequencing characterizes isoforms in thousands of cerebellar cells (PacBio)
• Single-nuclei isoform RNA sequencing unlocks barcoded exon connectivity in frozen brain tissue (PacBio/ONT)
• Altered cell and RNA isoform diversity in aging Down Syndrome patients (PacBio)

Immune profiling

• High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes (ONT) 
• Single-cell isoform analysis in human immune cells(ONT) 

Spatial Gene Expression

• A spatially resolved brain region- and cell type-specific isoform atlas of the postnatal mouse brain (PacBio/ONT)
• The spatial landscape of gene expression isoform in tissue sections (ONT)

Products:

Single Cell Gene Expression, Single Cell Immune Profiling, Single Cell Multiome ATAC+GEX, Spatial Gene Expression for Fresh Frozen.