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

Reveal more biology with long nanopore sequencing reads

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

Missing biology

Figure 1. 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 expression analysis from single cells. (Figure adapted from Volden & Vollmers, Genome Biol. 23:47 (2022), and made available under Creative Commons license 4.0)

High correlations between UMIs and genes detected per barcode

Figure 2. Top row: high correlations were observed for UMIs and genes detected per barcode between nanopore single-cell sequencing data and that of a short-read sequencing technology. Bottom row: UMAP projections showing 3’ gene expression of peripheral blood mononuclear cells obtained from nanopore single-cell data, clustered by isoform expression. The PTPRC gene shows cell-population-specific expression of alternative transcript isoforms, which were not detectable with short-read data.

Ultra-rich transcriptomic data without compromise

Maximise the value of single-cell experiments with nanopore sequencing: gain everything you would with short reads, plus full-length transcripts for more in-depth analysis. As demonstrated in our partner application note with 10x Genomics, nanopore sequencing shows high correlation with gene expression data from short-read sequencing, while also revealing cell-type-specific alternative isoform usage missed by short–read technology (Figure 2).

View the 10x Genomics application note
Case study

Single-cell nanopore sequencing reveals complex heterogeneity in leukaemia

Thijssen et al. applied a novel nanopore single-cell omics approach to study clinical research samples from patients with progressive leukaemia who failed therapy with a targeted agent. Combining short-read with nanopore long-read targeted and whole-transcriptome sequencing, the team identified mutations and alternative transcripts in specific sub-clones of the tumour at relapse.

Sequencing workflow

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 Ligation 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.

View our single-cell sequencing workflowView the Knowledge Exchange: Getting started
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Single-cell sequencing of full-length transcripts

For single cell and spatial RNA sequencing, we recommend the following:


Ligation Sequencing Kit

Analysis: wf-single-cell


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