Single-cell sequencing
The analysis of genomic heterogeneity at the single cell level has provided new insights into many research areas, including cancer research, cell development and function, and immunology. However, the use of traditional short-read sequencing technology limits the ability to identify transcript abundance at the isoform level. Long nanopore sequencing reads resolves this challenge, enabling the sequencing of full-length transcripts to gain a deeper understanding of complex biology.
Discover more biology — gene and isoform expression, plus variant detection in a single experiment
Efficient workflow — three hours from cDNA to sequencing
Simple data analysis: EPI2ME workflow includes barcode & UMI demultiplexing — no short reads required
Single-cell, isoform-level characterisation of RNA transcripts
The use of short-read single-cell (scRNA-seq) methodologies misses 80% of most transcripts. This is because short-read sequencing requires full-length transcripts to be fragmented, and results in bias towards sequencing the 3' or 5' end of transcripts (Figure 1). While suitable for simple gene counting, this approach misses important biology that can only be derived from an intact cDNA molecule. Nanopore sequencing enables analysis of full length transcripts — revealing previously obscured biology, including: isoform diversity, alternative splicing, expressed variants, and transcripts for nonsense-mediated decay
Figure 1: The power of single-cell sequencing comes from the facility to pool, sequence, and subsequently identify transcripts from hundreds of individual cells. All transcripts derived from the same cell can be identified using a unique cell barcode (cellBC). A unique molecular identifier (UMI), added to the original transcripts prior to amplification, further enables the identification of PCR duplicates that could impact transcript quantification, and can be used to generate more accurate consensus sequences. Short-read sequencing technologies only read approximately 90 bp of transcript sequence, precluding the identification of transcript isoforms. In contrast, long nanopore sequencing reads can span complete transcripts enabling in-depth, isoform-level gene expression analysis from single cells.
Figure 2: With the ability to obtain full-length transcripts using nanopore sequencing, Lebrigand et al. demonstrated the Clathrin light chain A gene ('Clta') isoform expression switch occurring during neuronal maturation in the mouse brain, displayed here in t-SNE plots a-c. Isoform Clta-204 was highly expressed in mature neurons (a), whereas isoform Clta-206 showed a higher expression level in precursor cells (b). Figure 2c provides an overlay of Clta-204/Clta-206 isoform expression in the isolated neuronal cells. Figure adapted from Lebrigand et al. 2020.
Ultra-rich transcriptomic data without compromise
Single-cell transcriptomics reveals intercellular gene expression heterogeneity at a level that cannot be achieved from bulk-cell analyses alone. Isoform-level expression data obtained from nanopore sequencing of single-cell libraries can reveal cell-type-specific differences in transcript splicing that are undetectable in short-read single-cell transcriptomic data. This is particularly useful for identifying phenomena such as isoform switching during cell development. For example, Lebrigand et al. demonstrated that, in the mouse brain, the Clathrin light chain A gene (‘Clta’) undergoes isoform switching during neuronal maturation, which they suggested may fine-tune the role this protein plays in different developmental stages (Figure 2). In this study, the team identified a total of 76 genes with cell-type specific transcript usage.
View our single-cell sequencing protocol based on the work of Lebrigand et al.
Single-cell transcriptomics with Oxford Nanopore: Getting started
In the first of a two-part Knowledge Exchange series on single-cell sequencing with Oxford Nanopore, Carly Tyer (Applications Scientist, Oxford Nanopore Technologies) provided an overview of sample and library preparation for single-cell transcriptome sequencing with nanopore technology. Carly focused on the benefits of single-cell analysis with Oxford Nanopore sequencing, including the ability to generate full-length cDNA sequences for exploring splice isoforms, obtaining quantitative expression data for comparing transcriptome variation at the single-cell level, and the identification of novel transcripts.
Carly presented an overview of the 10x Genomics Next GEM single cell 3’ preparation kit and how to get started with the ‘Single-cell transcriptomics with cDNA prepared using 10x Genomics’ protocol that enables the generation of full-length cDNA reads.
During the second Knowledge Exchange, Single-cell transcriptomics with Oxford Nanopore: data analysis, John Beaulaurier (Genomic Applications Bioinformatics Manager, Oxford Nanopore Technologies) guided users through the process of demultiplexing and conducting preliminary analyses on sequencing data produced from the Oxford Nanopore single-cell analysis pipeline. These steps are performed using the Sockeye open-source bioinformatic pipeline, which can now be executed via the wf-single-cell workflow in EPI2ME Labs. This workflow is designed to recover cell barcode and UMI sequences from the nanopore reads, as well as generate some basic outputs describing the composition of the single-cell sequencing data.
View our workflow on single-cell transcriptomics
Our workflow provides information on how to generate, sequence, and analyse full-length barcoded cDNA from single cells, using a combination of 10x Genomics and Oxford Nanopore platforms.
How do I perform single-cell cDNA sequencing using nanopore technology?
Start with full-length cDNA prepared using 10x Genomics Next GEM Single Cell 3’, 5’ gene expression or Visium Spatial Kits. The library can then be prepared for nanopore sequencing using the cDNA-PCR Sequencing Kit. We then recommend sequencing on a PromethION Flow Cell. The typical output from a PromethION Flow Cell is ~80 M cell-assigned reads, enabling the analysis of up to ~8,000 cells per flow cell (depending on desired read depth per cell). Primary data analysis, including barcode and UMI demultiplexing, can be performed via our EPI2ME software using the wf-single-cell workflow.
Discover how to identify alternative transcript isoforms at single-cell resolution
This application note from 10x Genomics describes the preparation of full-length cDNA libraries for nanopore sequencing using the 10x Genomics' Chromium Single Cell and Visium Spatial Gene Expression assays. Libraries were sequenced on the Oxford Nanopore PromethION. It was found that cell calling and clustering from long nanopore sequencing reads was comparable to short-read data. Further analysis of nanopore data identified differentially expressed isoforms between cell types. Find out more by downloading the application note.
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