Transcriptome
Nanopore sequencing technology uniquely delivers high yields of long, full-length RNA, supporting quantification and complete transcriptome characterisation at the isoform level. Differential isoform expression and allele-specific affects are known to be important in the susceptibility to and progression of diseases (e.g. cancer), and individual drug responses. Nanopore sequencing allows the generation of novel insights into transcript isoform expression and usage across different experimental conditions and cells, including single-cell analysis.
Long-read sequencing of human tissues to study allelic effects on transcriptome structure
The advent of long read technologies for RNA-seq has the potential to transform transcriptome analysis, since it can directly measure full-length isoforms
Dafni Glinos, New York Genome Center, US
Oxford Nanopore sequencing
Traditional short-read technologies
Unrestricted read length
- Generate full-length transcripts — >20 kb single read transcripts demonstrated
- Unambiguously identify all splice variants
- Perform accurate allele-specific, isoform-level gene expression analysis — even at single-cell resolution
- Easily identify antisense transcripts and lncRNA isoforms
- Approximately 50-fold fewer reads required to cover the same number of transcripts
Read length typically 50–100 bp
The short reads generated by traditional RNA-Seq techniques only partially cover a transcript’s length, making it challenging to accurately assemble and quantify transcript isoforms. Short reads also exhibit high rates of multimapping, leading to data loss and reduced utility for genome or transcript annotation.
Direct, amplification-free protocols
- Analyse poly-A tail length and transcription start sites
- Detect base modifications (e.g. methylation) as standard — no additional prep required
- Eliminate amplification bias and read length limitations using the Direct RNA Sequencing Kit or direct cDNA sequencing protocol
Amplification required
The amplification requirement of traditional RNA-Seq approaches can introduce bias, reducing complexity of the total RNA pool and potentially causing increased abundance or drop-out of some RNA species.
Accessible and scalable
- Scalable devices to suit your needs — portable MinION starting at just $1,000, including sequencing reagents
- Flexible throughput with modular GridION and PromethION — run extensive time-course or treatment studies simultaneously
- Develop targeted assays using low cost Flongle
- Sample multiplexing — reduce costs and maximise experimental efficiency
Less accessible
Platform costs and infrastructure requirements can limit global accessibility. Sample batching may be required for optimal efficiency, potentially delaying results.
Streamlined workflows
- Sample prep in <3 hours with just 13 minutes of hands-on time (cDNA-PCR Kit)
- Choice of two unique kits: cDNA-PCR and direct RNA — minimising bias and providing flexibility in speed, yield, and identification of epigenetic modifications
Laborious workflows
Typical sample prep times of ~7 hours with ~3 hours of hands-on time.
Real-time data streaming
- Stop sequencing when sufficient data generated — wash and reuse flow cell
- Immediate access to results
Fixed run time with bulk data delivery
Increased time-to-result and inability to identify workflow errors until it’s too late, plus additional complexities of handling large volumes of bulk data all at once.
The value of full-length transcripts without bias
Discover more about how long nanopore RNA sequencing reads enable unambiguous isoform-level gene expression studies and how direct sequencing further eliminates bias and, with direct RNA sequencing, supports simultaneous detection of base modifications. Read case studies covering a wide variety of research areas, including how nanopore RNA sequencing revealed 9 of the top 10 expressed isoforms of the CACNA1C gene (a therapeutic target for neuropsychiatric disease) to be novel transcripts.
Get more transcriptomics content, including getting started guides, workflows, and videos, in our Resource centre.
Revealing isoform-level differential gene expression in hummingbirds
The hummingbird, which exhibits an incredible ability to utilise ingested sugars and release energy from lipids, provides an excellent system to study how high blood glucose levels can be maintained without negative effects to health. Using nanopore sequencing to study the hummingbird transcriptome, Ariel Gershman and colleagues at Johns Hopkins University, USA, identified approximately twice as many transcripts than obtained using a short-read sequencing technology. Furthermore, the team demonstrated how full-length sequencing reads enabled the identification of tissue-specific gene expression at the isoform level, offering potential new insights to minimise the impact of high glucose levels on human health.
With our long reads it becomes much easier to assemble these transcripts and to tell which transcripts are expressed for which gene
Ariel Gershman, Johns Hopkins University, US
Direct RNA sequencing of mouse brain samples from the RIKEN Aging project
At NCM 2022, Callum Parr (RIKEN Institute, Japan) presented his ongoing work investigating the role of RNA splicing on the brain during aging. He described how he will leverage long-read direct RNA nanopore sequencing to overcome current sequencing limitations and map the dynamic nature of RNA splicing in different brain cell types. The aims of this work will be to discover isoforms and RNA modifications involved in the ageing process and age-associated diseases, such as Alzheimer’s disease.
Scalable transcriptome sequencing
From powerful, portable Flongle and MinION devices to the high-throughput, high-output benchtop GridION and PromethION platforms — scale your RNA sequencing to match your specific research requirements.
* Theoretical max output (TMO). Assumes system is run for 72 hours (or 16 hours for Flongle) at 420 bases / second. Actual output varies according to library type, run conditions, etc. TMO noted may not be available for all applications or all chemistries.
GridION
Running up to five independent MinION or Flongle Flow Cells with powerful, integrated compute, GridION provides the flexibility to run multiple RNA and DNA sequencing experiments, on-demand — ideal for differential gene expression studies and busy labs.
With up to 48 independently addressable, high-capacity flow cells, PromethION 48 is ideal for busy labs wishing to combine de novo genome sequencing with transcript annotation, allele-specific expression studies, and rare isoform discovery.
Flexible, high-throughput sequencing with up to 24 independent, high-capacity flow cells — complete genomic and transcriptomic characterisation of large sample numbers.
Offering two independent high-output PromethION Flow Cells – ideal for confident quantification and qualification of full-length transcripts, including rare isoforms.
Analyse transcript isoforms and differential gene expression from just $1,000, including sequencing reagents.
All the benefits of nanopore sequencing in a powerful, fully-integrated handheld device.
Adapting MinION and GridION for smaller, routine tests and analyses, including isoform characterisation.
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