Information
This kit is recommended for users who:
- Want to achieve modal raw read accuracy of Q20+ (99%) and above
- Want to obtain duplex data
- Want to optimise their sequencing experiment for accuracy and output
- Require control over read length
- Would like to utilise upstream processes such as size selection or whole genome amplification
Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the "3rd Party Materials" tab for more detail.
The Ligation Sequencing Kit V14 features:
The Ligation Sequencing Kit V14 offers a flexible method of preparing sequencing libraries from dsDNA (e.g. gDNA, cDNA or amplicons). The library preparation method is straightforward: DNA ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module before the sequencing adapters, supplied in the kit, are ligated onto the prepared ends.
The kit is optimised to achieve sequencing accuracies of over 99% (Q20+) with high output on our latest nanopore: R10.4.1. Our flow cell priming and sequencing reagents have been reformulated to be compatible with this improved Kit 14 adapter and R10.4.1 nanopore.
Kit 14 also includes previous updates such as the higher capture rate of DNA to enable lower flow cell loading amounts, and fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.
Users can either start with 1 μg of gDNA or 100-200 fmol of shorter-fragment input such as amplicons or cDNA. If your experiment requires long reads, it is recommended to start with full-length gDNA, and fragmentation/shearing is not advised. Please note: if long reads are required, high molecular weight DNA should be used as input. To determine purity, we suggest using the Nanodrop to measure the A260/280 and A260/230 ratios and we recommend that the sample should meet the following criteria:
- A260/280 = 1.8
- A260/230 = 2.0-2.2
The Ligation Sequencing Kit V14 is compatible with upstream processes such as target enrichment by sequence capture, whole genome amplification (for applications where under 1 ng of sample is available) and size selection (for enrichment of specified fragment lengths, using our Short Fragment Eliminator kit (EXP-SFE001)).
A) Read lengths observed when HMW gDNA was prepared using the Ligation Sequencing Kit with and without size selection (EXP-SFE001 treatment). B) Read N50 observed under the same sequencing conditions, demonstrating significant increase when size selected using EXP-SFE001.
PCR- and WGA-free workflows remove amplification bias and retain base modification information, which can be analysed using tools supported by Oxford Nanopore.
Further considerations:
Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and can reduce sequencing output. PCR can be used to generate more input material in cases where sample amount is limiting.
Shipping and logistics:
Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.