Oxford Nanopore Delivers Technology Update At Annual London Calling Conference: Bringing Together Years of Innovation to Showcase One Sensing Platform for All Biological Analyses
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 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 early access 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. 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: o 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. 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. Proof of concept data generated on Flongle sensor chip, prototype now being built: A new generation of nanopore, 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 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. 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.
