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.
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).
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.
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.
View our single-cell sequencing protocol based on the work of Lebrigand et al.
Knowledge exchange
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.
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.
Sequencing workflow
How do I prepare single-cell cDNA for sequencing using nanopore technology?
Single-cell gene expression can be captured using the 10x Genomics microfluidics-based Chromium platforms, which produce full-length, barcoded cDNA when using the protocol for the 10x Genomics Next GEM Single Cell 3’, 5’ gene expression or Visium Spatial Kits. The sequencing library can then be prepared using the Oxford Nanopore cDNA-PCR sequencing kit. It is possible to generate high read counts of full-length transcripts from 5,000 or more individual cells on a single PromethION Flow Cell. The typical output expected from a PromethION Flow Cell is ~80 M cell-assigned reads, out of ~150 M total reads. For more information, including analysis options, we recommend referring to our workflow and the resources listed in the ‘Related resources’ section below.
Application note
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.
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:
Find out about the versatile Flongle platform.
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