Q20+ Chemistry
Q20+ Chemistry for single molecule accuracy of 99% and higher
The Kit 14 chemistry for Q20+ raw read accuracy consists of an updated sequencing enzyme, in combination with the new R10.4.1 nanopore. This chemistry provides the highest accuracy for nanopore sequencing on measures such as SNP and INDEL scores, along with gold-standard performance for 5-methylC . A high-accuracy, high-output chemistry, it is capable of operating in three run conditions:
- Accuracy
- Default
- Output
Each run condition, set through the operating software MinKNOW, passes DNA through the pore at a distinct translocation speed, balancing requirements for higher accuracy or greater yields as required — whilst delivering simplex reads of Q20 or higher. All conditions are compatible with duplex reads, with data from both strands of the DNA molecule combined for a single molecule accuracy ~Q30 or higher. The duplex approach has already demonstrated perfect reads up to 72 kb in length, and Q30 reads at 260 kb.
Kit 14 chemistry simplifies the kit offering from Oxford Nanopore, ensuring users need not make upstream choices between highest output or highest accuracy when choosing how to go about answering their biological question.
Specific applications
Whilst Kit 14 chemistry is rolled out, some applications are better served by alternative solutions. Follow the links below for more details
RNA sequencing
Optimised for simplicity of cDNA preparation or direct RNA sequencing, read more about our RNA sequencing offerings.
SARS-CoV-2 sequencing
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.
16S sequencing
Multiplex kits for microbial 16S sequencing and genus-level bacterial identification
Sequencing in the field
Remove the reliance on cold-chain and leave the lab with our lypholised field sequencing kits
Available now
Oxford Nanopore Kit V14 chemistry enables a range of sample preparation approaches, with more kit functionality being released over time:
- Ligation Sequencing Kit V14 (SQK-LSK114) and Ligation Sequencing Kit XL V14 in open early access
- Native Barcoding Kit 96 V14 and Native Barcoding Kit 24 V14 (for 96 and 24 samples respectively) in open early access
- Rapid chemistry barcoding Kit 14 options – coming soon
- Ultra-long and Amplification kit options – coming 2022
Find out more on the release phases for Oxford Nanopore products, including early access phase
See how Q20+ is being used by the Community
Miten Jain: Human genome assembly and analysis using R10.4.1 and Kit 14
Miten Jain's team at University of California, Santa Cruz, USA have been assessing recent improvements in read length, accuracy, and software for ultra-rapid, nanopore-based whole-genome sequencing analysis. Specifically focusing upon Kit 14 here, Miten presented data generated in the past few days prior to this talk, showing the generation of over 140 Gb of sequencing data in a single PromethION Flow Cell. This high output is achieved with lower DNA and library input requirements than for the previous chemistry. Also highlighted was a notable improvement in raw read accuracy, particularly evident in homopolymer regions where lengths of up to approximately 20 bases were accurately called.
The team is currently generating their first human genome assemblies using the latest nanopore chemistry and flow cells; however initial results using sheared genomic DNA, are reaching NG50 scores exceeding 20 Mb. They are also evaluating the use of duplex data and developing further variant calling and methylation analysis tools. With the developments presented here, Miten concluded that “phased, Q40+ accuracy de novo assemblies may be achievable”.
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'.
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
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