Ligation Sequencing Kits enable ligation-based, PCR-free nanopore sequencing of native DNA.

• Sequence native DNA — eliminate PCR bias and retain base modifications
• Median raw read accuracy of Q20+ (99%) and above
• Optimized for accuracy and output
• Obtain duplex data
• Control over read length
• Multiplex samples with native barcoding kits

Ligation Sequencing Kits offer a flexible method of preparing sequencing libraries from dsDNA (e.g. gDNA, cDNA, or amplicons) for nanopore sequencing. A range of Ligation Sequencing Kits are available to suit your experimental requirements:

Ligation Sequencing Kit (SQK-LSK114) – the latest nanopore sequencing chemistry, optimised to achieve sequencing accuracies of over 99% (Q20+) when combined with R10.4.1 flow cells (FLO-MIN114). This kit is available in three formats: Ligation Sequencing Kit (SQK-LSK114) – for singleplex samples; Native Barcoding Kit 24 (SQK-NBD114.24) – standalone kit enabling PCR-free multiplexing of up to 24 samples per run; and Native Barcoding Kit 96 (SQK-NBD114.96) – standalone kit enabling PCR-free multiplexing of up to 96 samples per run. Note: These kits are compatible with R10.4.1 flow cells only.

The library preparation method is straightforward: DNA ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then sequencing adapters, supplied in the kit, are ligated onto the prepared ends. With no requirement for amplification, these kits eliminate potential PCR bias and preserve base modifications, which can be detected alongside the nucleotide sequence.

For highest data yields, we recommend starting with 100 to 200 fmol of pure input DNA for singleplex samples. Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and can reduce sequencing throughput.

Ligation Sequencing Kits are 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 the BluePippin, for example). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.

PCR- and WGA-free workflows remove amplification bias and retain base modification information, which can be analysed using tools supported by Oxford Nanopore.

The Ligation Sequencing Kit (SQK-LSK114) and the Native Barcoding Kits (SQK-NBD114.24 / SQK-NBD114.96) are compatible with R10.4.1 Flow Cells only.