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Single-cell sequencing

The analysis of genomic and transcriptomic 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 can introduce limitations in single-cell assays; for example, in transcriptome studies, it is not possible to identify transcript abundance at the isoform level. Long nanopore sequencing reads resolve these challenges, enabling end-to-end sequencing of full-length transcripts and large genomic regions in single reads, and spanning repetitive regions and structural variants.

Sequence full-length RNA transcripts for isoform-level single cell transcriptomics

Sequence full-length RNA transcripts for isoform-level single cell transcriptomics

Characterise splicing, chimeric transcripts, and sequence diversity across entire molecules

Characterise splicing, chimeric transcripts, and sequence diversity across entire molecules

Scale to your requirements with a range of nanopore sequencing platforms

Scale to your requirements with a range of nanopore sequencing platforms

Introduction

Introduction

Single-cell, isoform-level characterisation of RNA transcripts

Short-read based single-cell RNA sequencing (scRNA-Seq) methodologies only yield information from a small region close to one end of the transcript, precluding the facility to analyse splicing, chimeric transcripts, and sequence diversity across the molecule. In contrast, nanopore technology, which has no requirement for fragmentation, nor read length limitations, sequences the entire RNA (cDNA) molecule. As a result, full-length transcripts can be sequenced in single reads, allowing accurate, isoform-level characterisation and quantification (Figure 1).

How barcoding UMI's work

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 50 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.

Introduction 2
tSNE plot of single-cell clusters

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.

Full-length single-cell cDNA data reveals gene expression heterogeneity

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.

Case study

CASE STUDY

Comprehensive characterization of single-cell full-length isoforms in human and mouse with long-read sequencing

‘…because we have full-length isoform information, we are not just restricted to only looking for mutations in the final exon’

Matthew Ritchie

In the single-cell breakout session at the Nanopore Community Meeting 2020, Matthew Ritchie (The Walter and Eliza Hall Institute of Medical Research, Melbourne) presented a single-cell sample preparation method to generate full-length transcripts for nanopore sequencing ('FLT-seq') and an analysis tool for exploring isoform expression heterogeneity at the single-cell level ('FLAMES'). With these two methods his team explored differential transcript usage in single cells from various sample types, and identified variants with distinctive distributions between cell clusters. 

Within this session, presentations were also given by Rebecca Berrens (University of Cambridge, UK), who described her work investigating transposable element expression in single cells using nanopore technology, and Christoph Dieterich (University Hospital Heidelberg, Germany), who presented the ScNapBar software for processing and analysing single-cell data.

Sequencing workflow
PromethION with kits and flow cells
Interested in both single-cell and spatial transcriptomics?

Discover how the subtleties of cellular diversity can be revealed with a combination of single-cell and spatial transcriptomics approaches.

Sequencing workflow

How do I prepare single-cell cDNA for sequencing using nanopore technology?

The most common single-cell sample preparation methods use emulsions to isolate single cells and generate full-length cDNA; however, the cDNA is often subsequently fragmented prior to sequencing. Methods have been developed within the nanopore user community which skip the cDNA fragmentation step and allow preparation of full-length cDNA libraries for nanopore sequencing, using the Ligation Sequencing Kit for library preparation. With this approach, Lebrigand et al. have demonstrated that sequencing a full-length cDNA single-cell library on a single PromethION Flow Cell typically generates ~50 million cell and UMI-assigned reads. For more information, including analysis options, we recommend referring to the publication by Lebrigand et al., and others listed in the ‘Related resources’ section below.

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GET STARTED

Single-cell transcriptomics of human and animal cells

To obtain high yields of human or animal single-cell transcriptome data, we recommend the following:

portable MinION
Looking to QC your library first?

Find out about the versatile Flongle platform.

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