Ligation Sequencing Kit V14

A versatile sequencing kit optimised for modal raw read accuracy of Q20+ (99%+) and long read singleplex samples.

This uses our latest Kit 14 chemistry

  • Products from Oxford Nanopore Technologies that have completed their Early Access
  • Users can continue to provide feedback through feature request pinboard
  • Warranty periods extended to 1 – 3 months
  • Product continues to iterate with upgrade notifications of up to 3 months
  • Lead time visible in store
  • Product subject to availability


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:

Preparation time~60 minutes
Input requirement1 µg of gDNA or 100-200 fmol of amplicons or cDNA
PCR RequiredNo
FragmentationOptional; recommended for inputs of 100-500 ng
Kit chemistryKit 14 (V14)
Read lengthEqual to fragment length
Associated protocolsLigation Sequencing gDNA (SQK-LSK114)
Ligation Sequencing Amplicons (SQK-LSK114)
Ligation Sequencing gDNA - lambda control (SQK-LSK114)
Ligation sequencing gDNA V14 - low input by PCR (SQK-LSK114 with EXP-PCA001)
Ligation sequencing V14 - Direct cDNA sequencing (SQK-LSK114)
Ligation sequencing gDNA V14 - reduced representation methylation sequencing (RRMS) of human samples (SQK-LSK114)
Ligation Sequencing gDNA V14 - human sample (N50 30 kb) on PromethION (SQK-LSK114)
Ligation sequencing gDNA V14 - human sample (N50 10 kb) on PromethION (SQK-LSK114)
Ligation sequencing gDNA V14 - whole genome amplification (SQK-LSK114)
Ligation sequencing V14 - Single-cell transcriptomics with cDNA prepared using 10X Genomics on PromethION (SQK-LSK114)
Multiplexing alternatives• Native Barcoding Kit 24 V14 (SQK-NBD114.24)
• Native Barcoding Kit 96 V14 (SQK-NBD114.96)
• Ligation sequencing V14 - PCR Barcoding (SQK-LSK114 with EXP-PBC001 or EXP-PBC096)
• Multiplex Ligation Sequencing Kit XL V14 (SQK-MLK114.96-XL)
Pack size6 reactions
StabilityShipped at 2–8°C

Long-term storage -20°C

Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer

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.

Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.

The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.


The Ligation Sequencing Kit 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 FFPE repaired and end-prepped/dA-tailed using the NEBNext End Repair/dA-tailing module, and then sequencing adapters, supplied in the kit, are ligated onto the prepared ends.

LSK110 workflow

What's in the box

The Ligation Sequencing Kit V14 contains sufficient reagents to generate six sequencing libraries.

SQK-LSK114 v3

NameAcronymCap colourNo. of vialsFill volume per vial (µl)
DNA CSDCSYellow135
Ligation AdapterLAGreen140
AMPure XP BeadsAXPAmber11,200
Ligation BufferLNBWhite1200
Long Fragment BufferLFBOrange21,800
Short Fragment BufferSFBClear21,800
Sequencing BufferSBRed1700
Elution BufferEBBlack11,200
Library BeadsLIBPink1600
Library SolutionLISWhite cap, pink label1600
Flow Cell FlushFCFClear cap, light blue label18,000
Flow Cell TetherFCTPurple1200

Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

3rd party materials


  • NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (cat # E7180S). Alternatively, you can use the three NEBNext® products below:
  • NEBNext FFPE Repair Mix (M6630)
  • NEBNext Ultra II End repair/dA-tailing Module (E7546)
  • NEBNext Quick Ligation Module (E6056)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 80% ethanol in nuclease-free water
  • Qubit™ Assay Tubes (ThermoFisher Q32856)
  • Qubit dsDNA HS Assay Kit
  • (Optional) Bovine Serum Albumin (BSA) (50 mg/mL) (e.g Invitrogen™ UltraPure™ BSA (50 mg/mL) cat. # AM2616)


  • Hula mixer (gentle rotator mixer)
  • Magnetic separator, suitable for 1.5 ml Eppendorf tubes
  • Microfuge
  • Vortex mixer
  • Thermal cycler
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Ice bucket with ice
  • Timer
  • Qubit fluorometer (or equivalent for QC check)

Optional equipment

  • Agilent Bioanalyzer (or equivalent)
  • Eppendorf 5424 centrifuge (or equivalent)


The Ligation Sequencing Kit V14 can be used together with:


  • Flow Cell Wash Kit (EXP-WSH004)
  • Control Expansion (EXP-CTL001)
  • Flow Cell Priming Kit (EXP-FLP004)
  • Flow Cell Priming Kit XL (EXP-FLP004-XL)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
  • PCR Barcoding Expansion 1-12 (EXP-PBC001)
  • PCR Barcoding Expansion 1-96 (EXP-PBC096)
  • PCR Expansion (EXP-PCA001)
  • LFB Expansion (EXP-LFB001)
  • SFB Expanion (EXP-SFB001)

Flow cells

  • FLO-MIN114
  • FLO-PRO114M
  • FLO-FLG114
  • FLO-MIN114HD
  • FLO-PRO114HD


  • Flongle
  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION