Oxford Nanopore technology updates show consolidations and updates for a single, high-performance sequencing platform
At the Nanopore Community Meeting, Rosemary Sinclair Dokos, Stuart Reid and Lakmal Jayasinghe showed how the Oxford Nanopore sequencing platform’s capabilities continue to be pushed. They provided a summary of the current platform, imminent platform developments, and the innovation pipeline. Multiple updates and consolidations to improve and simplify the platform were presented, showcasing a single, simple, high-performance sequencing technology.
The latest R10.4.1 flow cells in combination with Kit 14, for high-yield, high-accuracy (Q20+ single molecule) sequencing, are expected to move from ‘early access’ to ‘released’ status in the first quarter of 2023. This enables all users to generate highly accurate, information-rich data, in real time and from any length fragments of DNA/RNA, providing one platform for all users’ sequencing needs.
Updates were provided that included developments in platform chemistry, hardware, data analysis, preparation and product range:
Current platform status and imminent developments
- Comprehensive, information-rich genomics
- Low cost, high-yield accessible sequencing
- Simple, versatile and consistent platform for end-to-end analysis
- Simple pathways for assay developers: Project TurBOT and Q-Line
- Unique platform features
Oxford Nanopore sequencing today, and imminent developments
Comprehensive, information-rich genomics
Oxford Nanopore’s sequencing platform provides a comprehensive view of human, and any other genomes, with simple, scalable workflows. This is driven by the ability to sequence any-length DNA/RNA fragments and to sequence native molecules to elucidate methylation for free and in real time. Updates that enhance this include:
- Sequencing with high-yield and high-accuracy Kit 14, in combination with R10.4.1 flow cells, will move from early access to general release in Q1 2023
- The standard 400 bases per second (bps) conditions delivers >Q20 (>99%) single molecule, single-pass accuracy at high yields
- The chemistry provides complete, continuous, accurate genomes, delivering a comprehensive picture of genomic variation from push-button analysis workflows
- Simplex data – from single strands of DNA – is showing Q20+ (>99%) single molecule accuracy and, comprehensive high performance across all human genetic variation including SNP, SNV and methylation
- Duplex sequencing – from complementary strands of DNA for higher accuracy – is now achieving Q30 (>99.9%) accuracy for more challenging applications. Enhanced rates of duplex data within runs can now be seen, for example >70% duplex rate was shown from an E.coli dataset. Flow cells are currently offered under a developer release
- New compute performance: with the Dorado basecaller and NVIDIA’s PromethION A100 processor, high accuracy basecalling (HAC) results are available in real time during the 72-hour sequencing run. Dorado is expected to be introduced into the operating software, MinKNOW, in Q1 2023
- Base modifications provide critical insights into many biological questions, including cancer research. Oxford Nanopore provides the most accurate and comprehensive technique for base modifications, offering 99.5% raw read accuracy for 5mC calling (2-way classification)
- The Kit 14 and R10.4.1 model for 5mC base modification is available and integrated now
- 5mC and 5hmC model is available as a stand-alone now, with a goal of integrating into MinKNOW in Q1 2023 offering users unprecedented insights into methylation of their samples
- Upgrades are largely delivered through consumables and software, so that users do not need to invest their research funds in new devices.
Low cost, high-yield, accessible sequencing
Users can access ultra-high yield sequencing data at competitive pricing. At 1-2 human genomes per PromethION Flow Cell today, this provides customers with the ability to sequence up to 9,984 whole human genomes per year at 30x coverage from $345 per genome, including library preparation costs. Further chemistry developments are in the pipeline designed to further extend the yield of PromethION Flow Cells.
Simple, versatile and consistent platform for end-to-end analysis
The Oxford Nanopore platform is versatile and can be tailored for a range of use cases, including whole genome sequencing, epigenetic analysis, transcriptomic and single cell approaches, and applications requiring both short (down to 20 bases) and ultra-long (over 4 million bases) fragments to be sequenced. Real-time data permits users to generate the answers they need, when they need them, even with targeted sequencing.
From sample preparation to analysis, platform improvements were shown that better support users with end-to-end analyses:
- To support consistent high-yield performance and a new wave of PromethION users on the new PromethION 2 (P2), new end-to-end workflows are being introduced, initially focusing on human samples (cells, blood), which are supported by the expanding Oxford Nanopore field application scientist team. Users should expect to routinely achieve >100Gb per PromethION Flow Cell using these protocols with R10.4.1 flow cells and Kit 14
- Protocols are being released that enable sample recovery after sequencing, designed to support users with particularly rare/precious samples so that they can drive for maximum data per sample, rather than per flow cell
- The EPI2ME analysis platform provides simple, powerful analytical pipelines at the push of a button, from a comprehensive picture of human variation to metagenomics to single cell. EPI2ME labs enables local or distributed sequencing and GitHub for user defined workflows and analysis. An open data analysis ecosystem has now been created, to combine user workflows with the Oxford Nanopore platform. Users can run their own analysis pipelines from EPI2ME with simple point and click implementation. Register your interest here
- Users can develop their own workflows in GitHub, increasing the reach of the simplicity of the product.
Enabling simpler pathways for assay developers: Project TurBOT and Q-Line
- Oxford Nanopore has been investing in a range of ISO9001-qualified devices with locked-down, standardised format, that will integrate Q20+ chemistries in a controlled offering with fewer and more predictable releases with a refreshed launch of ‘Q-Line’
- Project TurBOT integrates standard high sample-throughput liquid handling devices with sequencing devices such as MinION and P2 Solo to deliver sample preparation and analysis in a single device. Register your interest here
Unique platform features
Adaptive sampling, where users can use the software, MinKNOW, to select and enrich regions of interest for targeted sequencing, is a unique capability unlocked by real-time sequencing. This ‘intelligent’ method is being used in many ways, including focusing in on cancer genes in a human genome, or enriching pathogen DNA in a broader human/metagenomic sample.
- Adaptive sampling is already built into MinKNOW, and has now been demonstrated on P2
Updating small, portable formats
MinION Mk1D is in development – the next generation of portable sequencing that is designed to be compatible with the new iPad Pro®. With USB-C connectivity, the Mk1D can utilise the power of M1/M2 Apple® silicon for basecalling whilst also combining simple sequencing with the market leading user interface. Those interested can now register their interest in Mk1D here.
Pipeline developments: Towards sequencing by anyone, anywhere
Oxford Nanopore integrates accessibility into its product design, in order to broaden access to genomics.
- Research teams have been utilising machine learning to develop new enzymes that pass DNA through a nanopore faster. With greater speeds these have the potential to deliver 400Gb of sequence data, and as many as four human genomes from a single PromethION Flow Cell
- Additional chemistry and basecalling developments aim to characterise more diverse base modifications including 8oxoG and ribo-bases in DNA
- New nanopores are in development with almost double the length of reader head of R10.4,1 with a view to further improved homopolymer resolution
- Only Oxford Nanopore offers direct RNA sequencing, including characterisation of base modifications. With new RNA-specific nanopores and faster enzymes, a threefold improvement in output with accuracy was shown, increased to 96%. In addition, an order of magnitude reduction in required sample input was shown, to 50ng. This is expected to be offered as a developer release early in 2023
- Oxford Nanopore seeks to redefine the term “liquid biopsy”, showing R&D progress that offers the promise of sequencing native DNA/RNA directly from complex biological fluids – without the need for lab equipment – with the goal of broadening access to this important technique
- A new method continues to be developed, with the goal of delivering unparalleled single molecule accuracy, including for low frequency (eg below 0.1%) genetic variants. By combining standard sequencing with ‘Outy’, a method of reversing the direction of sequencing through a single nanopore, it is possible to re-read a single molecule forwards-and-backwards through that nanopore. This can continue as many times as is required to reach the desired accuracy. Base modifications are also detected with high-accuracy by the Outy method.
London Calling 2023
Next year’s London Calling will be 17th – 19th May, early bird tickets will be available soon!