At NCM, announcements include single-read accuracy of 99.1% on new chemistry and sequencing a record 10 Tb in a single PromethION run
Thu 3rd December 2020
During an update at the 2020 Nanopore Community Meeting, Oxford Nanopore revealed a number of key advances in its sequencing technology. These include the release of new improvements in PromethION flow cells that have enabled a new 10 Terabase sequencing record, modal single-read accuracy of 99.1% using a new sequencing chemistry currently in development, high accuracy variant calling tools and automation options for nanopore sequencing experiments at any scale.
Further accuracy improvements
A new analysis algorithm, Bonito CRF, was recently released by Oxford Nanopore, that provides increased accuracy single read basecalling – the process of identifying the sequence of bases on an individual molecule of DNA. This latest update to Bonito builds on previous work to deliver improved performance, and is trained with a larger, more diverse data set. Using Bonito CRF, Oxford Nanopore has shown 98.3% single read accuracy on the current chemistry, and nanopore customers have also reported >98% single read accuracy.
High single read accuracy supports very high consensus accuracy (where a molecule is sequenced multiple times); optimised analysis tools including Guppy/Bonito basecalling, assembly with Canu/Flye and polishing with Medaka, can now enable Q45 with R9.4.1 flow cells and Q50 with R10.3 flow cells.
Variant calling performance is also improving with the latest releases; structural variation (SV) accuracy has reached gold standard of 96% with 30X rather than 60X coverage. SNP accuracy is now at 99.92%, which is comparable to short read accuracy.
In addition, this week Oxford Nanopore has generated modal raw-read accuracy of 99.1% (99%=Q20) using a new chemistry with Bonito, delivered on internal validation sets with a substantial fraction of these raw reads above Q20.
A new yield record
In November, Oxford Nanopore started to ship PromethION flow cells that incorporated a number of iterative improvements in yields and consistency. These improvements have culminated in a 10 Tb internal run using all 48 Flow Cells, a 25% improvement on the previous record. The median output per Flow Cell in this run was 208 Gb — the equivalent of two human genomes over 30X coverage. This success has been reflected in customer data, with increasing yields reported across a range of applications.
Further improvements on the PromethION platform are expected with the release of R10.3 PromethION flow cells in the coming weeks.
Automated sample preparation, at scale
As the number of users of nanopore sequencing in production-scale environments increases, Oxford Nanopore has been focusing during 2020 on automation solutions that enable users to run high numbers of samples, to run larger projects, or simply to automate everyday workflows.
Oxford Nanopore is collaborating with Hamilton to combine expertise to automate library preparation workflows on Hamilton’s NGS STAR 96. The first protocol released through the collaboration enables preparation of 96 samples in under 5 hours, with only one hour of hands-on time. One run fills two PromethION 48s, enabling high yield, highly validated sample prep and sequencing output.
Oxford Nanopore is also working with Opentrons to deliver mid-range lab automation that is suitable for users running the MinION or GridION devices. Opentrons automation liquid handlers are accessible, compact, and can support 2,500 LamPORE tests a day or automation of the Ligation kit, and ARTIC native barcoding method, as well as being suitable for future PCR based applications.
On the small scale level VolTRAX has seen a number of developments this year, including the development of a PCR-ready cartridge design which will be available soon, alongside the existing Sequencing and Multiplex Kits.
Additional updates from the talk:
- In early 2020, nanopore sequencing flow cells using the R10.3 nanopore, for improved homopolymer resolution and improved methylation analysis, were released for the MinION and GridION devices. R10.3 flow cells for use with the high-throughput PromethION device, or for rapid, low cost sequencing on the Flongle adapter, will be commercially available imminently.
- Datasets: To support a better understanding of the latest nanopore tools and sequencing data, Oxford Nanopore has released a human genome data set, HG002. This demonstrates the Oxford Nanopore sequencing platform’s latest performance, and will be regularly updated as new novel algorithms, tools and chemistries are developed.
- Epigenetics: Following continuous innovations that drive an improved ability to characterise methylated bases during the sequencing process, nanopore sequencing now gives strong correlation to whole genome bisulphite sequencing, even at low read depth of ~20X. The analysis delivers a higher number of methylated positions called, better reproducibility than bisulphite sequencing, and is available as part of the standard nanopore sequencing process with no additional sample preparation or cost.
- Sequencing long fragments: Oxford Nanopore devices continue to enable the direct sequencing of any length fragment of DNA/RNA, from short to ultra-long; the current record for longest fragment sequenced is 4.2 Mb. Sequencing longer fragments provides rich data on structural variants, which account for the largest number of divergent base pairs across human genomes and contribute to multiple human diseases.
- The release of ‘Adaptive sampling’ on GridION enables real-time, intelligent target enrichment with no upfront sample preparation. A user programmes the MinKNOW software to target specific regions of interest. Nanopore sequencing then enriches for these regions in real time, by rejecting reads from non-target fragments. This method retains read length and methylation information, for rich discovery.
- A Cas9 sequencing kit was recently launched for targeted nanopore sequencing. This kit enables high depth sequencing of previously inaccessible target regions capturing read lengths of up to 100 kb, retaining methylation information.
Oxford Nanopore outlined a pipeline of R&D, including:
- New manufacturing methods to target very low cost Flongle flow cells, to open up frequent, rapid, low cost nanopore analyses
- The ongoing development of a voltage sensing platform to dramatically increase density of nanopore sensing; if these channels are engineered at a 40um pitch, this has the potential to deliver ~100,000 nanopore channels on a piece of silicon 16x30mm, with the onward potential to generate an estimated 3.9Tb of sequence data in a day