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New nanopore sequencing chemistry in developers’  hands; set to deliver Q20+ (99%+) “raw read” accuracy  

Mon 1st March 2021

A new nanopore sequencing kit is now being tested by a number of developers in the nanopore user community.  The kit features a modified enzyme, tweaked run conditions and a further improved basecalling model in the Bonito basecaller. These improvements have produced raw single-molecule, single-pass sequencing reads above a modal 99% accuracy, or Q20+ in external users’ hands, as well as internally.   The improved software also allows optional filtering of lower-quality reads, moving the mean accuracy closer to Q20 without sacrificing read length.  

This novel chemistry, as first mentioned at the Nanopore Community Meeting in December, makes use of Oxford Nanopore’s existing electronic nanopore sensing platform to deliver improved accuracy on an individual read (“raw read”) basis.   

Oxford Nanopore recently released Bonito v0.3.6 — an updated basecaller that has been shown to boost performance to a modal raw accuracy of 98.3% with existing in-field sequencing chemistry. This basecaller update can also be used to re-analyse older data and significantly improve it.

Oxford Nanopore’s R&D teams are currently working on additional methods to drive further improvement of single-molecule basecalling over the course of 2021.   This includes a potential multi-pass chemistry designed to yield highly accurate single-molecule consensus reads, without sacrificing read length. Work has also advanced on native calling of natural base modifications at the single molecule level.  Further updates on this pipeline will be provided in due course.

New chemistry

An early version of the “Q20+” kit is now in use by initial developer sites around the world, who are currently providing feedback that will inform further development of the final kit and basecalling algorithms.  Oxford Nanopore intends to broaden the early access phase in due course and will provide updates in the coming weeks.

“It’s great to see that we can replicate the performance seen by Oxford Nanopore of the new Q20+ chemistry,” said Professor Matt Loose, from DeepSeq at the University of Nottingham. “The yield is good and pore occupancy is fantastic. We're looking forward to trying this chemistry on other samples to better measure the improvements to assembly and consensus accuracy for our more challenging projects very soon.”

“I find it pretty amazing that even this early access version of the kit can produce good throughput highly accurate single pass data within ~1day of sequencing a complex metagenome sample.” said Rasmus Kirkegaard, University of Vienna, “I am sure raw read Q20 any length sequencing will be a game changer!”.

Alignment accuracy overall

Image: Rasmus Kirkegaard, University of Vienna

Nanopore sequencing uniquely combines a number of properties, including sequencing DNA or RNA fragments of any length, real-time data generation and retention of base modifications, which alongside very high single molecule accuracy gives rich data about the underlying biology in a sample.   

Very high single molecule accuracy supports the generation of high-quality consensus sequences, where the molecule is read multiple times, allowing for the generation of reference-quality genomes.   

Oxford Nanopore has driven continuous improvement in accuracy of its sequencing platform since launch, through combining new sequencing chemistries with continuously evolving data analysis algorithms.  In addition to high single-pass accuracy, the nanopore platform is now able to deliver single molecule consensus accuracy of 99.995% with UMIs, and Q50 (99.999%) consensus sequencing data. Nanopore sequencing outperforms traditional short read technologies on its ability to elucidate structural variants, and can provide SNV calling accuracy of 99.92 (F1) on the current R9.4 flow cells. For further information on the tool sets that support these performances please visit the nanopore sequencing accuracy page

Higher PromethION yields drive cost efficiencies for high throughput sequencing  

In addition to developments on accuracy, Oxford Nanopore announced at NCM in December 2020, the release of upgraded PromethION flow cells, that are driving consistently higher yields in users hands.  This has supported a recent in-field record of 245Gb from a single PromethION flow cell, or the equivalent of nearly three 30X human genomes on a single flow cell.  Noting that a PromethION can run up to 48 flow cells, and that PromethION flow cells can be purchased for as little as $625, this development supports ultra-high throughput nanopore sequencing.  To find out more about PromethION, visit the website.

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