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
Characterise splicing, chimeric transcripts, and sequence diversity across entire molecules
Scale to your requirements with a range of nanopore sequencing platforms
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).
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 isoform usage.
High throughput, error-corrected nanopore single-cell transcriptome sequencing
‘…combining the high throughput of nanopore sequencing with UMI guided error correction allows both high confidence definition of transcript isoforms and identification of sequence heterogeneity in single cells.’
Lebrigand et al. demonstrated how the addition of long nanopore sequencing reads to traditional short-read single-cell sequencing approaches enabled the accurate analysis of full-length transcripts. Following cell isolation using 10x Genomics technology, 1,141 cells from an E19 mouse brain library, across two batches, were subject to both long-read nanopore sequencing on the PromethION device and short-read sequencing. The short-read data was used to identify the cell barcode (cellBC) for each transcript and the associated unique molecular identifiers (UMIs). These data were then used to support the assignment of cellBC and UMIs to the genome-aligned nanopore sequencing reads.
In total, the team identified 18,439 previously-annotated transcripts and 15,317 novel transcripts, demonstrating the enhanced power of incorporating long sequencing reads to single-cell transcriptome studies. The data enabled the segregation of different cell types and the identification of differential isoform expression between cell types. Furthermore, the full-length reads provided more detailed insight into the association of individual SNVs in the glutamate receptor Gria2, and the identification of sequence heterogeneity within and between cell types.
Discover how the subtleties of cellular diversity can be revealed with a combination of single-cell and spatial transcriptomics approaches.
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.