RNA and cDNA sequencing
Without direct RNA sequencing, we would have to perform a series of different experiments to obtain this data … now we can get all this information from a single experiment
Josie Gleeson, University of Melbourne, Australia
Identify, characterise, and quantify full-length transcripts at the isoform level — up to single-cell resolution
Analyse poly-A tail length and explore epigenetic modifications, such as methylation, through direct RNA sequencing
Deliver accurate gene expression data with fewer reads than legacy short-read sequencing platforms
Characterise whole transcriptomes
Nanopore technology uniquely delivers high outputs of any-length reads that span full-length RNA transcripts, facilitating their accurate, unambiguous analysis and resulting in complete transcriptome characterisation at the isoform level. Nanopore RNA and cDNA sequencing generates novel insights into differential isoform expression and allele-specific expression, even at single-cell resolution. Furthermore, with direct RNA sequencing that does not require reverse transcription or amplification, it is possible to detect base modifications (such as methylation) in native RNA transcripts. The scalability of nanopore sequencing devices means that researchers do not need to batch a high number of samples, reducing waiting times and generating faster results.
Featured content
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The value of full-length transcripts without bias
Discover how nanopore sequencing enables unambiguous, full-length, isoform-level gene expression analysis, including identification, characterisation, and quantification. See how researchers use direct native RNA sequencing to simultaneously detect base modifications, such as methylation, in a single experiment.
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Detecting isoforms and RNA modifications with nanopore sequencing
In this best-practice, end-to-end workflow, discover how to characterise full-length splice isoforms and RNA modifications with direct RNA nanopore sequencing.
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PromethION 2 Solo and 2 Integrated
Offering the flexibility of two independent, high-output PromethION Flow Cells, the compact PromethION 2 devices bring the benefits of high-coverage, real-time nanopore sequencing to every lab. Ideal for low-cost access to highly accurate whole transcriptomes.
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Compact benchtop device designed to run and analyse up to five MinION Flow Cells.
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A flexible, large-scale, direct DNA and RNA sequencing device — delivering on-demand access to terabases of data.
Technology comparison
Oxford Nanopore sequencing
Legacy short-read sequencing
Any read length (50 bp to >4 Mb)
Short read length (<300 bp)
- Generate complete, high-quality genomes with fewer contigs and simplify de novo assembly
- Resolve genomic regions inaccessible to short reads, including complex structural variants (SVs) and repeats
- Analyse long-range haplotypes, accurately phase single nucleotide variants (SNVs) and base modifications, and identify parent-of-origin effects
- Sequence short DNA fragments, such as amplicons and cell-free DNA (cfDNA)
- Sequence and quantify full-length transcripts to annotate genomes, fully characterise isoforms, and analyse gene expression — including at single-cell resolution
- Resolve mobile genetic elements — including plasmids and transposons — to generate critical genomic insights
- Enhance taxonomic resolution using full-length reads of informative loci, such as the entire 16S gene
- Assembly contiguity is reduced and complex computational analyses are required to infer results
- Complex genomic regions such as SVs and repeat elements typically cannot be sequenced in single reads (e.g. transposons, gene duplications, and prophage sequences)
- Transcript analysis is limited to gene-level expression data
- Important genetic information is missed
Direct sequencing of native DNA/RNA
Amplification required
- Eliminate amplification- and GC-bias, along with read length limitations, and access genomic regions that are difficult to amplify
- Detect epigenetic modifications, such as methylation, as standard — no additional, time-consuming sample prep required
- Create cost-effective, amplification-free, targeted panels with adaptive sampling to detect SVs, repeats, SNVs, and methylation in a single assay
- Amplification is often required and can introduce bias
- Base modifications are removed, necessitating additional sample prep, sequencing runs, and expense
- Uniformity of coverage is reduced, resulting in assembly gaps
Real-time data streaming
Fixed run time with bulk data delivery
- Analyse data as it is generated for immediate access to actionable results
- Stop sequencing when sufficient data is obtained — wash and reuse flow cell
- Combine real-time data streaming with intuitive, real-time EPI2ME data analysis workflows for deeper insights
- Time to result is increased
- Workflow errors cannot be identified until it is too late
- Additional complexities of handling large volumes of bulk data
Accessible and affordable sequencing
Constrained to centralised labs
- Sequence on demand with flexible end-to-end workflows that suit your throughput needs
- Sequence at sample source, even in the most extreme or remote environments, with the portable MinION device — minimise potential sample degradation caused by storage and shipping
- Scale up with modular GridION and PromethION devices — suitable for high-output, high-throughput sequencing to generate ultra-rich data
- Sequence as and when needed using low-cost, independently addressable flow cells — no sample batching needed
- Use sample barcodes to multiplex samples on a single flow cell
- Bulky, expensive devices that require substantial site infrastructure — use is restricted to well-resourced, centralised locations, limiting global accessibility
- High sample batching is required for optimal efficiency, delaying time to results
Streamlined, automatable workflows
Laborious workflows
- Prepare samples in as little as 10 minutes, including multiplexing
- Use end-to-end whole-genome, metagenomic, targeted (including 16S barcoding), direct RNA and cDNA sequencing workflows
- Scale and automate your workflows to suit your sequencing needs
- Perform real-time enrichment of single targets or panels without additional wet-lab prep by using adaptive sampling
- Lengthy sample prep is required
- Long sequencing run times
- Workflow efficiency is reduced, and time to result is increased
