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Oxford Nanopore Touts Tech Improvements Including PromethIon, Accuracy, Automation

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NEW YORK – Oxford Nanopore Technologies this week announced a number of product releases, technology improvements, and development projects during its Nanopore Community Meeting, held online this year.

According to James Clarke, VP of platform technology, the firm has made several improvements to its PromethIon platform. "It's probably been our best six months on that platform," he said.

For a start, the firm has overcome a number of manufacturing problems with the PromethIon chips that affected the connectivity to the ASIC and the structure of the microwells. "The good news is that those things have been fixed," Clarke said, and as a result, sequencing yields at PromethIon customer sites have been going up.

The company has also reworked the synthetic membrane that holds the nanopores in its sequencing chips, he said, improving its robustness and consistency. The new membrane is now part of sequencing chips for the PromethIon, GridIon, MinIon, and Flongle sequencers, which should result in fewer membrane-related sequencing failures, he said.

Company researchers have also improved the nanopore insertion method for the PromethIon chips, increasing the number of pores by 20 to 25 percent, which should translate to greater data output. It rolled out the new chips a few weeks ago.

Internally, Oxford Nanopore has set a new record for PromethIon output, he said, achieving 10 terabases of data from a single instrument run with 48 flow cells using six different PCR-amplified human DNA samples. The median output per flow cell was 208 gigabases in this run and the maximum output for a single flow cell was 242 gigabases.

For users interested only in very long reads in the range of 100 kilobases, he said, the company has developed an ultra-long read kit to maximize those reads. The kit uses a DNA extraction and purification method developed in collaboration with Circulomics, though customers can also use other extraction methods, and it employs the company's transposase chemistry, which is gentler than its ligation-based chemistries that require the use of beads.

Using ultra-long read kit internally, company researchers have been able to get around 90 gigabases of data from a single PromethIon flow cell with an N50 read length of more than 100 kilobases, Clarke said, and have established a new record read length of 4.15 megabases, almost doubling the previous record.

Overall, PromethIon output varies depending on read length, he said: using libraries designed for 10 to 30 kilobase reads, users can expect about 220 gigabases of data per flow cell. Ultra-long read libraries for 100 kilobase reads or so will yield about 100 gigabases per flow cell and require four interactions with the instrument over a four-day run time. Customers can now also pool several samples per PromethIon flow cell using barcodes, for example two human genomes for 30X coverage or three human genomes for 20X coverage.

To improve output further, Oxford Nanopore is working on a new flow cell design with deeper wells that can take more mediator solution – needed for passing current through the pores – as well as on a way to increase the number of channels by about 10 percent, he said.

For the low-throughput Flongle, the company has been able to increase production and meet demand this summer, Clarke said, "and hopefully we can continue to do that as demand increases." Oxford Nanopore is also working on new methods to reduce production costs for Flongle flow cells to "a few dollars," he added.

Clarke also provided an update on a long-running R&D project that aims to replace today's current sensing with voltage sensing around the nanopore. This would allow the firm to pack the nanopores and underlying electronics much more tightly. "It is high risk, but it is very high reward," he said. The firm has made "more progress in the last six months" on this than ever before, he added, and expects to have a prototype ASIC toward the end of the year.

The company has also made further improvements to its data accuracy. According to Stuart Reid, VP of development, the modal raw read accuracy has now reached 98.3 percent using a new base caller, called Bonito. He also hinted at two new sequencing chemistries in development that can deliver single-pass raw read accuracy greater than 99 percent, though he provided no details or commercialization timelines.

Bonito is currently available on Github as a research version and is expected to be integrated into the MinKnow platform in February or March of next year. In addition, the company has made improvements to various tools and methods for consensus base calling, SNP calling, and structural variant calling.

The new base caller, combined with the ultra-long read kit, can yield "very respectable human assemblies" from 60X coverage data, Reid said. Adding the Pore-C assembly method has also allowed the company to generate "reference-quality assemblies from a very modest amount of data."

Oxford Nanopore has applied these methods to data from the telomere-to-telomere (T2T) consortium, he added, which sequenced the haploid cell line CHM13, and was able to generate more contiguous and accurate assemblies.

In addition, the company has developed a new cDNA sequencing kit, both for single and barcoded samples, which can generate more full-length transcripts. It is available to early-access customers at the moment and is expected to launch in the first quarter of next year.

In terms of methylation sequencing, the firm has found that its new R10.3 nanopores seem to be better at finding methylation signals than the previous R9.4.1 pores, Reid noted.

Adaptive sampling, where a DNA molecule is sequenced for a little while and users can decide on the fly whether they want to continue sequencing or move on to a new DNA strand, has now been released for the GridIon as a version that is fully integrated with the MinKnow platform. This will be followed by integrated releases for the Mk 1C and, later on, the PromethIon.

Rosemary Dokos, VP of product management, mentioned several developments for library prep automation. For example, the firm has released protocols for the Hamilton NGS Star liquid handler in conjunction with a kit called Ligation Kit XL that can prepare 96 samples in under 5 hours.

It has also been working with Opentrons, a maker of small automation solutions, on an automated workflow for its LamPore COVID-19 assay, which is CE-marked and runs on the GridIon Q-Line instrument. This involves an $18,000 Opentrons liquid handler, she said, and would allow 2,500 LamPore assays to be run per day. "We're very excited about the protocols that we will develop together in the future" with Opentrons, she said. Oxford Nanopore is also working on Q-versions for clinical and diagnostic use of the MinIon and the Mk 1C.

Finally, the VolTrax sample prep device has also evolved. The company has developed a PCR-ready cartridge that it plans to roll out next year, Dokos said, which will require existing VolTrax users to upgrade their devices.

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