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Oxford Nanopore TechnologiesFully scalable, real-time DNA/RNA sequencing technology
Oxford Nanopore DiagnosticsLamPORE – rapid, low-cost, scalable detection of SARS-CoV-2
London Calling 2022
18th - 20th May
Ligation Sequencing Kit (Q20+)
SQK-LSK112

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

This is an Early Access product

$599.00
2Early Access
  • Access the latest products from Oxford Nanopore Technologies
  • Provide feedback into the innovation team as the product develops
  • Product Warranty may initially be limited to 1 month as it is so new
  • Product routinely iterates as new features and capabilities are added
  • Estimated lead time in store
  • Product subject to availability

Information

This kit is recommended for users who:

  • want to achieve raw read sequencing modal accuracy of 99% and above (Q20+)
  • want to optimise their sequencing experiment for accuracy
  • require control over read length
  • would like to utilise upstream processes such as size selection or whole genome amplification.

This is an Early Access product
For more information about our Early Access programmes, please see this article on product release phases.

The Ligation Sequencing Kit features:

FeatureProperty
Preparation time~60 minutes
Input requirement1 µg of double-stranded gDNA or amplicons
PCR RequiredNo
FragmentationOptional; recommended for inputs of 100-500 ng
Read lengthEqual to fragment length
Read type produced1D
Associated protocolsLigation sequencing gDNA (SQK-LSK112)
Ligation sequencing amplicons (SQK-LSK112)
Ligation sequencing gDNA - Lambda control (SQK-LSK112)
Ligation sequencing gDNA - whole genome amplification (SQK-LSK112)
Ligation sequencing amplicons - PCR barcoding (SQK-LSK112 with EXP-PBC096)
Ligation sequencing amplicons - PCR barcoding (SQK-LSK112 with EXP-PBC001)
Ligation sequencing gDNA - low input by PCR (SQK-LSK112 with EXP-PCA001)
Ligation sequencing gDNA - PCR barcoding (SQK-LSK112 with EXP-PBC096)
Ligation sequencing gDNA - PCR barcoding (SQK-LSK112 with EXP-PBC001)
Multiplexing options• Flow Cell Wash Kit (EXP-WSH004)
• Native Barcoding Kit 24 (SQK-NBD112.24)
• Native Barcoding Kit 96 (SQK-NBD112.96)
• Native Barcoding Expansion (EXP-NBD112)
Pack sizeSix 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 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, and then sequencing adapters, supplied in the kit, are ligated onto the prepared ends.

The kit is optimised to achieve sequencing accuracies of over 99% (Q20+). For highest data yields, we recommend starting with 50-100 fmol of pure input DNA. Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and can reduce sequencing output.

The Ligation Sequencing Kit (Kit 12) includes the Adapter Mix H (AMX H) with three key features: Firstly, the adapter is loaded with an updated sequencing enzyme enabling accuracies of over 99% (Q20+). Secondly, the adapter is higher capture, enabling lower flow cell loading amounts. Finally, the adapter contains the fuel fix technology enabling users to run long experiments without the need for fuel addition during the run.

Users can either start with 1 μg of gDNA or 50 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 neither advised nor required. 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 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 the BluePippin, for example). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.

Ligation fragmentation comparison The relative sequencing yields (A) and typical read-length histograms (B, C and D) observed when preparing E.coli libraries using the Ligation Sequencing Kit without or with g-TUBE fragmentation or with size selection (without fragmentation).

PCR- and WGA-free workflows remove amplification bias and retain base modification information, which can be analysed using tools developed in the Nanopore Community.

Further considerations:

Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and can reduce sequencing throughput. 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.

Workflow

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 contains sufficient reagents to generate six sequencing libraries.

SQK-LSK112

NameAcronymCap colourNo. of vialsFill volume per vial (µl)
DNA CSDCSYellow135
Adapter Mix HAMX HGreen240
AMPure XP BeadsAXPAmber11,200
Ligation BufferLNBClear1200
L Fragment BufferLFBOrange21,800
S Fragment BufferSFBClear21,800
Sequencing Buffer IISBIIRed1500
Elution BufferEBBlack11,200
Loading Beads IILBIIPink1360
Loading SolutionLSWhite cap, pink sticker on label1400
Flush BufferFBBlue61,170
Flush TetherFLTPurple1200

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.

3rd party materials

Consumables

  • 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 70% ethanol in nuclease-free water
  • Qubit™ Assay Tubes (ThermoFisher Q32856)
  • Qubit dsDNA HS Assay Kit

Equipment

  • 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)

Compatibility

The Ligation Sequencing Kit can be used together with:

Kits

  • Sequencing Auxiliary Vials (EXP-AUX002)
  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MIN106D
  • FLO-MIN112
  • FLO-PRO002
  • FLO-PRO112

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION