Oxford Nanopore delivers technology update at annual London Calling conference: bringing together years of innovation to showcase one sensing platform for all biological analyses
Thu 19th May 2022
At the annual London Calling conference, CTO Clive Brown and colleagues James Clarke, Stuart Reid, Lakmal Jayasinghe and Rosemary Sinclair Dokos have provided an Oxford Nanopore tech update summarising the status of its nanopore sensing platform, key recent developments that drive improved accuracy and increased output, and outlined the vision for future developments.
With the latest updates, nanopore sequencing can now deliver very accurate, comprehensive data outputs, at high yields, with the ability to sequence short or long fragments, in real time and capture all variant types in one experiment, on one platform. Oxford Nanopore technology enables users to fit the technology to their biological question.
Users are now able to perform high-throughput, comprehensive genomic, transcriptomic and epigenetic analyses on a single platform. Early data on the recognition of individual amino acids on our nanopore platform was also shared.
Highlights of the talk included:
- The introduction of “best of both' sequencing chemistry, combining the latest R10.4.1 flow cells with the latest Kit14 to deliver high-accuracy, high-output sequencing data, achieving 99.6% accurate single molecule raw-read simplex data and 99.92% duplex with tunable runs for further flexibility and optimisation;
- Current R9 flow cells and Kits 10/11 – already enabling a broad range of high-impact scientific analyses - will continue to be supported as users transition to R10.4.1 and Kit 14;
- First developer shipment of the palm-size PromethION 2 (P2) solo device, a high-output, affordable nanopore sequencer;
- Developments in methylation analysis tools, including Remora, making Oxford Nanopore the most comprehensive technology for characterising methylation;
- Examples of the utility of Short Fragment Mode on the nanopore platform, including methylation and nucleosome analysis of cell-free DNA;
- A new sequencing file format, pod5, that is designed to replace fast5 and enable faster file writing. This supports increasing device outputs and accuracy, enabling smaller raw data file sizes and streamlining downstream analysis;
- Ability to characterise individual amino acids in a short peptide sequence on the Oxford Nanopore platform; an early step towards protein sequencing.
- Accuracy and output
- Platform technology and devices
- Real time methylation analysis
- Any-length DNA/RNA fragments
- Protein analysis
Oxford Nanopore’s move to the R10 nanopore series continues to drive accuracy enhancements, alongside the new Kit14, launching this quarter. The move to R10 and Kit14 combines the best of the previous capabilities: very high accuracy (Kit12 includes newer enzymes but is slower) and outputs (Kit 9/10/11 have higher speeds but lower accuracy). Early community evidence is showing a strong performance with the new kit and flow cells.
- These chemistries can now be
tuned by modifying the run temperatures, which will be programmable in the software to:
- 260 bps (accuracy mode), 400 bps (default mode) 520 bps (output mode)
- With these settings, users can
achieve modal simplex raw read accuracies of:
- 99.6% (accuracy mode), 99.2% (default mode) 99% (output mode)
- All tunable run settings deliver duplex accuracies around Q30 (99.9%) with the accuracy mode delivering 99.92% duplex accuracy.
- With the newest chemistry, the longest duplex read that perfectly aligns to a human reference is 72 Kb, and longest Q40 read 144 Kb.
With the output mode (520 bps), the teams at Oxford Nanopore have achieved 307 Gb on a single PromethION Flow Cell.
Variant calling accuracy continues to improve with further chemistry enhancements. Full details can be found at www.nanoporetech.com/accuracy and they will be available in EPI2ME labs during May 2022.
Platform technology and devices
As Oxford Nanopore continues to open up DNA sequencing to anyone, anywhere, it has now launched a first-in-class handheld, low-cost, ultra-high-throughput DNA sequencing device. The first PromethION 2 (P2) solo has been shipped and can deliver high-output, low-cost sequencing in a palm-sized device. Designed to make high-throughput sequencing more accessible, the P2 supports users wishing to discover more, rich biological insights about human disease, including cancer and ultra-long plant and animal genomes or larger transcriptome/metagenomic analyses. This new device can produce as much as 580 Gb sequence data in one run.
In further device updates, Oxford Nanopore discussed the development of the MinION Mk1D. This is designed to be an accessory keyboard with an integrated sequencer for tablet devices and will mean that users can simply dock a recommended tablet into the MinION Mk1D, pair it, and initiate sequencing.
Specifications for all devices can be found at the specification table https://nanoporetech.com/products/specifications
Multiple routes to platform improvement include flow cell design, lower sample volumes, membrane enhancements, integration of sample/library prep, longer run times through no need to flush, and manufacturing process improvement as Oxford Nanopore scales up to drive improving quality of product. Additional platform developments include:
- Oxford Nanopore is continuing development of the new low power ASIC, to drive further form factors for distributed analyses. Proof of concept data generated on Flongle sensor chip, prototype now being built
- A new generation of nanopore ("Rx"), even longer reader head than R10, shows promise in further homopolymer improvement, currently at simplex accuracy of 98%, having improved 8% since December; development continues.
- A new basecalling framework, Dorado has been introduced - that will ultimately speed up access for users. Dorado is designed with support for Apple GPUs and new NVIDIA hardware. It is also projected to keep up with high accuracy (HAC) models on new PromethION 48 hardware.
- Adaptive sampling, already fully available on GridION, is now in beta release on PromethION and MinION Mk1C and will be fully released soon. This can achieve 5-10X target enrichment, depending on the target
Real-time methylation analysis
Nanopore sequencing of native DNA/RNA enables users to gain information about base modifications alongside nucleotide sequence. The recent release of Remora – Oxford Nanopore’s latest methylation analysis tool - further enhances base-modification analysis on nanopore devices. Remora will be integrated into MinKNOW next week, enabling much simpler access to methylation data in real time.
Nanopore sequencing can differentiate between modifications such as 5mC and 5hmC and novel modifications can be trained into the algorithms as these become fully characterised, ensuring a complete methylation picture from a single experiment, significantly more methylation events than detected by bisulphite and with a simpler experimental process. Remora 1.0 models improve signal scaling which results in higher detection accuracy and quality filtered calls achieving 99.8% accuracy for 5mC in CpG contexts
This means that Oxford Nanopore is now the most comprehensive technology for characterising methylation, with our first releases aimed at targeting all CpG areas.
Any-length DNA/RNA fragments
Oxford Nanopore’s technology sequences DNA or RNA molecules of any length, from short to ultra-long. It is the only technology on the market capable of sequencing DNA lengths spanning five orders of magnitude in a single technology.
Short Fragment Mode (SFM) is now available on the device operating software, MinKNOW, and is designed to enable nanopore sequencing of fragments as short as 20 bases. Oxford Nanopore has demonstrated more than 250M native human reads, with an average read length ~ 200 bases, on a PromethION Flow Cell, opening up many potential uses where shorter fragments need analysis, at scale and at a competitive cost, but while retaining features such as real time methylation analysis.
Following the analysis of CTO Clive Brown’s own genome and cell-free DNA isolated from plasma — the ‘cf Cliveome’ dataset has been released. This uses SFM, which has uncovered interesting biology, including the potential for tissue typing from methylation data and greater insight into nucleosome arrangements. It was noted that 28M CpG methylation sites on short fragments are accessible to nanopore sequencing in the human genome, compared to 850K in current arrays. The Cliveome cfDNA sequence dataset can be accessed here.
Oxford Nanopore continues to develop and provide a range of kits to enable a broad range of analyses:
- The release of Kit 14 will roll out through Oxford Nanopore ligation kits, including native barcoding kits; 384 barcoding versions are now in development. Following the ligation range the rapid range of kits will be upgraded to enable Q20+ accuracies with simple 10 minutes library preparation.
- Short fragment eliminator: this kit to support long fragment libraries is scheduled for release into early access in June
- Ultra-long DNA Sequencing Kits are being upgraded to Kit14 chemistry and are with developers now
- cDNA sequencing kits can provide 160M reads on PromethION and 20M on MinION, for cost-effective transcriptome analysis that supports expression analysis and full-length transcripts, gene fusions, isoform discovery, cell sub type, spatial single cell genomics; the single cell analysis pipeline is now on Github
- The direct RNA protocol now on the Nanopore Community reduces input volumes.
Oxford Nanopore R&D teams have shown on MinION that the platform can be used to characterise amino acids in a short peptide sequence. This is an early step towards developing protein sequencing capabilities on the nanopore platform and further work will include further optimisation of chemistry to achieve better protein signals and development of analytical methods to predict characteristics of unknown peptides.
Years of innovation are now being integrated towards a simpler but versatile platform, to provide high performance analyses across genomics, transcriptomics, epigenetics, and with a pathway towards proteomics.
- Platform: James Clarke, VP Platform Technology
- Chemistry: Lakmal Jayasinghe, VP R&D Biologics
- Data: Stuart Reid, VP Development
- Product: Rosemary Dokos, VP Product Management
- New and notable: Clive Brown, CTO
This announcement contains certain forward-looking statements. Phrases such as "potential", “expect”, "intend", “believe we can”, “working to”, "anticipate", "when validated", and similar expressions of a future or forward-looking nature should also be considered forward-looking statements. Forward-looking statements address our expected future business, and by definition address matters that are, to different degrees, uncertain and may involve factors beyond our control.