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Ligation Sequencing Kit XL (Q20+)
SQK-LSK112-XL

A versatile sequencing kit optimised for modal raw read accuracy of Q20+ (99%+) and long reads. Multiple samples can be processed simultaneously.

This is an Early Access product

This product has a 6 week lead time

$4,000.00

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
  • would like to process multiple samples simultaneously, either with a multichannel pipette or liquid handling robot
  • 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 XL features:

FeatureProperty
Preparation time~120 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 protocolsGenomic DNA by Ligation
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 size48 reactions

The Ligation Sequencing Kit XL offers a flexible method of preparing sequencing libraries from dsDNA (e.g. gDNA, cDNA or amplicons). The larger kit allows users to process multiple samples simultaneously with a multichannel pipette or liquid handling robot. 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 XL (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 XL 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.

2018 05 30 LSK109 workflow v1 DS (1)

Protocols

Genomic DNA by Ligation (SQK-LSK112-XL)

To access the protocol, you will need to register for a Nanopore Community account.

What's in the box

The Ligation Sequencing Kit XL contains sufficient reagents to generate 48 sequencing libraries.

LSK112-XL tubes

NameAcronymCap colourNo. of vialsFill volume per vial (µl)
DNA CSDCSYellow1100
Adapter Mix HAMX HGreen1320
Ligation BufferLNBWhite11,500
L Fragment BufferLFBWhite cap, orange stripe on label220,000
S Fragment BufferSFBWhite cap, blue stripe on label220,000
Sequencing Buffer IISBIIRed31,500
Elution BufferEBWhite cap, black stripe on label110,000
Loading Beads IILBIIPink21,500
Loading SolutionLSWhite cap, pink sticker21,500
Flush BufferFBWhite cap, blue stripe on label415,500
Flush TetherFLTWhite cap, purple sticker11,600

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)
  • Agencourt AMPure XP beads
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with heat seals
  • 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

  • Magnetic separator suitable for 96-well PCR plates, e.g. DynaMag™-96 Side Skirted Magnet (Thermo Fisher, # 12027)
  • OR magnetic separator suitable for 0.2 ml PCR tube strips, e.g. DynaMag™-PCR Magnet (Thermo Fisher, #492025) or DynaMag™-96 Side Magnet (Thermo Fisher, #12331D)
  • Hula mixer (gentle rotator mixer)
  • Microfuge
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, #11766427)
  • 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
  • Pipetting troughs
  • Qubit fluorometer (or equivalent for QC check)

Optional equipment

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

Compatibility

The Ligation Sequencing Kit XL can be used together with:

Kits

  • Sequencing Auxiliary Vials (EXP-AUX110)
  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D
  • FLO-MIN112
  • FLO-PRO002
  • FLO-PRO112

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION