-
What is rapid-based sequencing?
Rapid-based chemistry is the method that the sequencing adapter is attached (rapid attachment) to the DNA ends. Typically, in the transposase step, the DNA is cut and the adapter is attached at the same time without any ligation enzymes. However, in the barcoding kits, the barcodes are attached during the transposase step and the sequencing adapters are attached in a later step using rapid attachment. It is also worth noting that in the PCR-based kits, adaptation is completed by PCR.
These kits are optimised for speed and simplicity or for applications where turnaround times are more critical or laboratory equipment is limited.
In comparison to the Ligation Sequencing Kit V14, the Rapid Sequencing Kit V14 sequencing output is slightly reduced due to the lack of purification steps and the presence of transposases; both of which may cause pore blockages, preventing the pore from sequencing adapted DNA (Figure 3). Read length is also slightly reduced due to the requisite transposase fragmentation.
As the Rapid Sequencing Kit focuses on a rapid library preparation, certain steps, such as bead purification, are omitted and may reduce sequencing yield in some cases. For further information on improving flow cell performance, please refer to the Flow cell performance section in the Sample input and recommendations section.
Figure 3. The graph illustrates typical output achieved with the Rapid Sequencing Kit compared to the Ligation Sequencing Kit.Our current rapid sequencing kits available are:
- Rapid Sequencing Kit V14 (SQK-RAD114)
- Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
- Rapid Barcoding Kit 96 (SQK-RBK110.96)
- Rapid Barcoding Kit V14 24 (SQK-RBK114.24)
- Rapid Barcoding Kit V14 96 (SQK-RBK114.96)
- Rapid PCR Barcoding Kit (SQK-RPB114.24)
- 16S Barcoding Kit 24 V14 (SQK-16S114.24)
- Midnight RT PCR Expansion (EXP-MRT001)
Below, we outline the sample input requirements and library preparation workflows for our simplex kits. For information on the barcoding kits, please see the Barcoding kits section.
-
Sample input recommendations
Before starting the Rapid Sequencing Kit library preparation, it is important to ensure that you are using the correct amount of starting material to ensure a successful sequencing experiment. After DNA extraction, we recommend quantifying your DNA samples:
Quantification Method Mass Qubit Fluorometer and Qubit dsDNA BR Assay Kit Size • Agilent 2100 Bioanalyser for samples of <10 kb
• Agilent FemtoPulse for samples of >10 kb
• Oxford Nanopore FlonglePurity Nanodrop 2000 Spectrophotometer For information regarding how to quantify the mass of DNA samples for library preparation input, please refer to the Sample input and recommendations section.
For our latest Kit 14 upgrade of the kit (SQK-RAD114), a reduced starting input of 100 ng of HMW gDNA containing long fragments is recommended. Using less than 100 ng or samples with shorter fragments can compromise sequencing output because yield from the library preparation will be reduced. Where only lower inputs are available, we recommend using the Rapid PCR Barcoding Kit 24 V14 (SQK-RPB114.24) to increase the number of template molecules.
Please refer to the Sample input and recommendations section of this document for further information about sample input recommendations and how to improve library quality.
Data output from the flow cell is influenced by the amount and quality of the input DNA sample fragmented by the fixed amount of transposase in the sequencing kit to generate tagged fragments.
- To generate long fragments:
- Add more than the recommended starting input as there will be fewer cuts per molecule
- Long fragments of DNA must be present initially in the sample input
- To generate short fragments:
- Add less than the recommended starting input as there will be more cuts per molecule
Due to the simple nature of the workflow and the fact that little sample manipulation is required (e.g. minimal pipetting steps and no clean-up steps) some very long reads can be achieved with this kit, despite the required transposase fragmentation. However, in order for long reads to be observed in sequencing, long fragments need to be present in the sample in the first place. However, to generate ultra-long reads, we recommend using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114), which uses rapid-based chemistry. Further information for this sequencing kit is available below.
- To generate long fragments:
-
Rapid Sequencing Kit V14
The Rapid Sequencing Kit V14 (SQK-RAD114) is our newest rapid-based sequencing kit optimised for speed and simplicity, using limited laboratory equipment, and a lower starting input of 100 ng of HMW gDNA. This kit has been upgraded to use our newest Kit 14 chemistry, which includes improved modal raw read sequencing accuracies with higher output on our latest nanopore: R10.4.1. This Kit 14 upgrade also includes updates such as the higher capture rate of DNA to enable lower flow cell loading amounts, and fuel fix technology. Note, due to the higher capture of the adapter, it is important to follow the flow cell loading recommendations in the protocols.
This sequencing kit and protocol is optimised for speed and simplicity, requiring 100 ng of HMW gDNA input. Due to the minimal sample manipulation, required, very long reads can be generated despite the requisite transposase fragmentation as long as there are very long DNA fragments present in the input sample. However, for ultra-long reads, we recommend using the Ultra-Long DNA Sequencing Kit V14.
Workflow:
The library preparation uses transposase-based fragmentation of the gDNA input whilst simultaneously tagging the fragmented template with transposase adapter sequences. Post-transposition, sequencing adapters are then attached to the transposase adapters in an enzyme-free reaction.
-
Ultra-Long DNA Sequencing Kit V14
This is our updated Ultra-Long DNA Sequencing kit using rapid-based Kit 14 chemistry to sequence ultra-long reads. The kit includes new reagents to simplify the library preparation and to improve DNA precipitation and recovery. This kit uses rapid-based chemistry due to the simple workflow, which enables very long reads to be achieved due to the minimal pipetting required during library preparation.
This kit requires ultra-high molecular weight (uHMW) gDNA to be extracted. In our protocol, we recommend using the NEB Monarch® HMW DNA Extraction Kit for Tissue (T3060) to extract the uHMW gDNA from either frozen cells or whole blood. After gDNA extraction, a diluted fragmentation mix containing transposases are added to the extracted gDNA to fragment and simultaneously tag the fragmented template with transposase adapter sequences. Post-transposition, sequencing adapters are then attached to the transposase adapters in an enzyme-free reaction. After an overnight elution with the Precipitation Star (PS), the library is ready for sequencing.