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
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, 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
