Gene expression
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'Our results show that current gene transcript annotations are incomplete and supports the use of long-read sequencing to identify novel RNA isoforms'
Ricardo De Paoli-Iseppi, The University of Melbourne, Australia
Accurately characterise and quantify full-length transcripts with nanopore reads of unrestricted length
Generate high sequencing yields from low input amounts
Eliminate PCR bias and explore epigenetic modifications with direct RNA sequencing
Full-length transcripts in single reads
Analysis of gene expression is important in many applications, from clinical research to developmental biology. However, the use of legacy short-read technologies can cause multi-mapping when aligning data and limit quantification accuracy; this can be further limited by PCR bias.
In contrast, Oxford Nanopore reads of unrestricted length allow transcripts to be sequenced end to end, enabling accurate quantification and complete characterisation of isoforms in a single dataset. Furthermore, direct RNA sequencing enables the simultaneous detection of epigenetic modifications and eliminates PCR bias.
Featured content
The value of full-length transcripts without bias
This white paper focuses on the facility of nanopore RNA and cDNA sequencing to tackle challenges in the areas of full-length transcript identification, isoform characterisation and quantification, and viral detection.
Direct RNA sequencing workflow
This workflow shares how direct RNA nanopore sequencing delivers full-length transcript sequencing, enabling quantitation of gene and isoform expression without bias and detection of RNA modifications.
Recommended device for gene expression studies
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PromethION 2 Integrated and 2 Solo
Gain flexible, high-output nanopore sequencing for every lab with the PromethION 2 devices. Generate high depth of coverage of whole transcriptomes on up to two individually addressable PromethION Flow Cells, enabling high-resolution gene expression analysis.
The PromethION 2 Integrated is a self-contained, benchtop device with powerful onboard compute, while the compact PromethION 2 Solo utilises resource within a GridION or other existing compute.
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
