Q20+ Chemistry for single molecule accuracy of 99% and higher
Oxford Nanopore has announced a new Kit 14 Chemistry for Q20+ sequencing, containing an updated sequencing enzyme compared to that within the Q20+ Early Access programme, enabling Simplex accuracies of 99.3%. In combination with the R10.4.1 nanopore, Kit 14 chemistry (which sequences at ~420 bps) supports the highest single molecule accuracies from nanopore sequencing, through either Simplex (1D raw read, Q20+) or Duplex data (basecalls for both strands of the single DNA molecule combined, ~Q30). This chemistry provides the highest nanopore sequencing performance on accuracy measures such as SNP and INDEL recall and precision, along with leading performance for 5-methylC when compared to bisulfite sequencing.
With the upcoming release of this new chemistry, Oxford Nanopore will be simplifying its offerings to ensure users do not need to make choices during sample preparation between the highest outputs or the highest accuracy. With R10.4.1 and Kit 14 chemistry, users will be able to adjust the temperature at which their experiment is conducted to generate the data they require — and be able to answer their biological question.
Some applications may still be served best through use of our established kit 10 chemistry
Optimised for simplicity of cDNA preparation or direct RNA sequencing, read more about our RNA sequencing offerings.
With support for the ARTIC protocol, and Midnight for a simple, rapid method of sequencing SARS-CoV-2 genomes at low cost per sample, learn what you need to get started with nanopore sequencing for the SARS-CoV-2 virus.
Multiplex kits for microbial 16S sequencing and genus-level bacterial identification
Sequencing in the field
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See how Q20+ is being used by the Community
Miten Jain: Nanopore sequencing updates using Q20+ and R10.4
Miten Jain’s team at University of California, Santa Cruz, USA have been working on increasing the read lengths obtained with nanopore sequencing, through use of ultra-high molecular weight DNA from human samples. This has lead to sequencing runs on PromethION flow cells generating ~1/3 of their data in read lengths of 100 kb+. They have applied this expertise in nanopore sequencing to their work with the latest Q20+ chemistries, which when combined with the R10.4 nanopore has provided ‘a median accuracy of 99% using the most recent HG002 data’. With the use of this chemistry the resolution of homopolymers has also ‘substantially improved’, which in turn Miten’s group has found to improve assembly contiguity and accuracy. Presented here are their SNV calling results from the Q20+ R10.4 data, using the PEPPER-Margin-DeepVariant pipeline and achieving F1-scores of 0.999
Mantas Sereika: Nanopore R10.4 enables near-perfect bacterial genomes
Mantas, of Aalborg University, Denmark, presents here R10.4 Q20+ nanopore sequence data on microbial mock community samples. With modal raw read accuracy of 99.1%, along with improved homopolymer calling compared to R9.4.1 data, he believes that with sufficient coverage and consensus polishing all homopolymers in the genome should be resolvable. This trend was continued in complex metagenomic samples, with DNA extracted from anaerobic digester sludge showing improved homopolymer calling compared to R9.4.1 - between homopolymer lengths of 8 and 10, or lower, ‘most of the homopolymers in the metagenome assembled genomes seemed to be called correctly’. Finishing up with comments around Duplex reads present in their mock community R10.4 Q20+ sequence data, plotting the raw read accuracies ‘basically broke our R script’ due to a modal Duplex read acccuracy 99.9% (Q30) accuracy. Read more in their pre-print on bioRxiv: https://doi.org/10.1101/2021.10.27.466057
Alexander Wittenberg: Accuracy improvements in crop genome assembly using the Q20+ chemistry
Alexander, a long-time staff member of KeyGene, Netherlands, covered how they are developing a comprehensive computational toolset to perform crop genome analysis 'on an unprecedented scale', including de novo assembly, variant detection, and data visualisation. The team at KeyGene have been evaluating the performance of the Q20+ chemistry and the R10 nanopore on several plant species, as well as the impact of utilising plant-trained basecalling models. The latter’s effects can be seen in their maize whole-genome sequencing data, with a 2.5% increase in raw-read accuracy being observed. The former was presented in results for lettuce, from which the in-house STL assembler showed the best assembly metrics, and also from melon. With the melon genome they carried out a world-first, Duplex-only assembly, and observed consensus accuracies higher than those of alternative long-read technologies, summarising that 'Duplex reads are really amazing'.