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Introduction to Kit 14 chemistry
Our latest Kit 14 chemistry is an upgrade across our product range to enable highly accurate and tuneable sequencing.
Upgrades include:
- New E8.2 motor protein in our V14 sequencing kits (e.g SQK-LSK114)
- New R10.4.1 flow cells
- Basecalling upgrades for simplex or duplex data
Kit 14 builds on our previous chemistry upgrades to combine high output with Q20+ accuracy. Duplex rates can also be achieved to further improve accuracy to Q30+.Note: For highest sequencing accuracies, ensure the super-accurate basecaller (SUP) is selected for your sequencing parameters during run set-up on MinKNOW.
Figure 1. Example data expected to be achieved using the Ligation Sequencing Kit V14 (SQK-LSK114) on a GridION Flow Cell. -
Introduction to duplex data
Our Kit 14 chemistry has been developed to reach sequencing accuracies of ~Q30 with duplex data and SUP basecalling. Duplex basecalling is the reading of both strands for even higher accuracy compared to our previous kits.
We offer two options for basecalling Kit 14 data with sequencing accuracies of 99% and above (Q20+):
- Simplex basecalling: The sequencing of a single strand. The template DNA strand passes through the nanopore and is basecalled. This is performed on MinKNOW.
- Duplex basecalling: The sequencing of both strands. The complement strand is read immediately after the template strand and the consensus basecall for both strands leads to a further increase in accuracy. This is performed on either Dorado.
Duplex reads do occur at a lower level naturally. However, our Kit 14 chemistry include upgrades in the motor protein and pore in the flow cells, which in combination work to increase second strand capture to improve duplex rates. For more information on basecalling duplex data, please see the "Basecalling Kit 14 duplex data" section.Below, we illustrate duplex sequencing at the pore level (Fig. 2) and we demonstrate what data can be achieved with duplex sequencing (Fig. 3).
Figure 2. The diagram above demonstrates how duplex reads are sequenced on the flow cell, the first strand is the template strand and the second is the complement strand.
Figure 3. This graph illustrates percentage of data in a duplex pair. Duplex data is presented for both template and complement strands, therefore, for this example, duplex rate is 21.4%.