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Ligation Sequencing Kit
SQK-LSK109

A ligation-based sequencing kit for processing singleplex samples. We recommend using our latest version: SQK-LSK114. All COVID-related projects will be supported indefinitely.

Includes a Flow Cell Priming Kit

$599.00
4Fully released
  • Products from Oxford Nanopore Technologies that have been available for some time
  • Users can continue to provide feedback through feature req
  • Warranty periods of 3 months or more * Product continues to iterate with change notifications of 3-6 months
  • Lead time visible in store
  • Product subject to availability

Information

This kit is recommended for users who:

  • want to optimise their sequencing experiment for throughput
  • 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 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 Ligation Sequencing Kit features:

FeatureProperty
Preparation time60 minutes
Input requirement1000 ng of gDNA
PCR RequiredNo
FragmentationOptional; recommended for inputs of 100-500 ng
Read lengthEqual to fragment length
Associated protocolsLigation sequencing gDNA (SQK-LSK109)
Ligation sequencing gDNA - Lambda control (SQK-LSK109)
Ligation sequencing amplicons (SQK-LSK109)
Ligation sequencing gDNA - low input by PCR (SQK-LSK109 with EXP-PCA001)
Ligation sequencing gDNA - native barcoding (SQK-LSK109 with EXP-NBD104 and EXP-NBD114)
Ligation sequencing amplicons - native barcoding (SQK-LSK109 with EXP-NBD104 and EXP-NBD114)
Ligation sequencing gDNA - native barcoding (SQK-LSK109 with EXP-NBD196)
Ligation sequencing amplicons - native barcoding (SQK-LSK109 with EXP-NBD196)
Ligation sequencing gDNA - PCR barcoding (SQK-LSK109 with EXP-PBC001)
Ligation sequencing amplicons - PCR barcoding (SQK-LSK109 with EXP-PBC001)
Ligation sequencing gDNA - PCR barcoding (SQK-LSK109 with EXP-PBC096)
Ligation sequencing amplicons - PCR barcoding (SQK-LSK109 with EXP-PBC096)
Ligation sequencing amplicons - dual barcoding (SQK-LSK109 with EXP-NBD104, EXP-NBD114, and EXP-PBC096)
Ligation sequencing gDNA - whole genome amplification (SQK-LSK109)
Ligation sequencing gDNA - exome enrichment (SQK-LSK109)
Ligation sequencing gDNA - sequence capture (SQK-LSK109)
Ligation sequencing amplicons - custom PCR UMI (SQK-LSK109)
PCR tiling of SARS-CoV-2 virus - classic protocol (SQK-LSK109 with EXP-NBD104, EXP-NBD114 or EXP-NBD196)
PCR tiling of SARS-CoV-2 virus - Eco protocol (SQK-LSK109 with EXP-NBD104, EXP-NBD114 or EXP-NBD196)
PCR tiling of SARS-CoV-2 virus - automated Hamilton NGS STAR 96 (SQK-LSK109 with EXP-NBD196)
Multiplexing options• Native Barcoding Expansion 1-12, 13-24 and 96
• PCR Barcoding Expansion 1-12 and 1-96
• Flow Cell Wash Kit
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 kit is optimised to generate maximum throughput. For highest data yields, we recommend starting with 100-200 fmol of pure input DNA. Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and can reduce sequencing throughput.

Users can either start with 1 μg of gDNA, quantified using the Qubit fluorometer, 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 neither advised nor required. Please note: if long reads are required, then long fragments must be present in the starting material. 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.

2018 05 29 LSK109 v1 DS

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. Also, where sample purity is compromised and library preparation efficiency may be reduced, PCR will select for molecules which have been successfully adapted, and generate more of them. The original impurity is lost or diluted. It is often observed that a sample which produces poor sequencing results can be improved by inclusion of PCR. However, if PCR is likely to be the main method of library preparation, we recommend the PCR Sequencing Kit, or Rapid PCR Barcoding Kit.

While any dsDNA can be used as a template for the Ligation Sequencing Kit, users planning to regularly sequence amplicons or cDNA, should consider specific kits with dedicated protocols which simplify the laboratory workflow and produce superior results. For amplicons, we recommend using our four-primer PCR protocol along with the PCR Sequencing Kit (or PCR Barcoding Kit for multiplexing amplicons), and for cDNA we recommend the cDNA-PCR Sequencing Kit or the Direct cDNA Sequencing Kit.

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.

2018 05 30 LSK109 workflow v1 DS

Protocols

Ligation sequencing gDNA (SQK-LSK109)
Ligation sequencing gDNA - Lambda control (SQK-LSK109)
Ligation sequencing amplicons (SQK-LSK109)
Ligation sequencing gDNA - low input by PCR (SQK-LSK109 with EXP-PCA001)
Ligation sequencing gDNA - native barcoding (SQK-LSK109 with EXP-NBD104 and EXP-NBD114)
Ligation sequencing amplicons - native barcoding (SQK-LSK109 with EXP-NBD104 and EXP-NBD114)
Ligation sequencing gDNA - native barcoding (SQK-LSK109 with EXP-NBD196)
Ligation sequencing amplicons - native barcoding (SQK-LSK109 with EXP-NBD196)
Ligation sequencing gDNA - PCR barcoding (SQK-LSK109 with EXP-PBC001)
Ligation sequencing amplicons - PCR barcoding (SQK-LSK109 with EXP-PBC001)
Ligation sequencing gDNA - PCR barcoding (SQK-LSK109 with EXP-PBC096)
Ligation sequencing amplicons - PCR barcoding (SQK-LSK109 with EXP-PBC096)
Ligation sequencing amplicons - dual barcoding (SQK-LSK109 with EXP-NBD104, EXP-NBD114, and EXP-PBC096)
Ligation sequencing gDNA - whole genome amplification (SQK-LSK109)
Ligation sequencing gDNA - exome enrichment (SQK-LSK109)
Ligation sequencing gDNA - sequence capture (SQK-LSK109)
Ligation sequencing amplicons - custom PCR UMI (SQK-LSK109)
PCR tiling of SARS-CoV-2 virus - classic protocol (SQK-LSK109 with EXP-NBD104, EXP-NBD114 or EXP-NBD196)
PCR tiling of SARS-CoV-2 virus - Eco protocol (SQK-LSK109 with EXP-NBD104, EXP-NBD114 or EXP-NBD196)
PCR tiling of SARS-CoV-2 virus - automated Hamilton NGS STAR 96 (SQK-LSK109 with EXP-NBD196)

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

What's in the box

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

SQK-LSK109 v3

NameAcronymCap colourNo. of vialsFill volume per vial (µl)
DNA CSDCSYellow150
Adapter MixAMXGreen140
Ligation BufferLNBClear1200
L Fragment BufferLFBWhite cap, orange stripe on label21,800
S Fragment BufferSFBGrey21,800
Sequencing BufferSQBRed2300
Elution BufferEBBlack1200
Loading BeadsLBPink1360
Sequencing TetherSQTPurple110

The Flow Cell Priming Kit is also supplied.

FLP

NameAcronymCap colourNo. of vialFill volume per vial (µl)
Flush BufferFBBlue61,170
Flush TetherFLTPurple1200

3rd party materials

Consumables

  • Agencourt AMPure XP beads
  • NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (E7180S)
  • Alternatively, you can purchase NEB reagents individually:
    • NEBNext FFPE Repair Mix (M6630)
    • NEBNext 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

Optional consumables

  • Covaris g-TUBE (for fragmenting input amounts between 100 and 500 ng)

Equipment

  • Hula mixer (gentle rotator mixer)
  • Magnetic rack
  • Microfuge
  • Vortex mixer
  • Thermal cycler at 20°C and 65°C
  • 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

Optional equipment

  • Agilent Bioanalyzer (or equivalent)
  • Qubit fluorometer (or equivalent for QC check)
  • Eppendorf 5424 centrifuge (or equivalent)

Additional materials for:

PCR barcoding (with the 12 or 96 barcoding expansion)

  • LongAmp Taq 2X Master Mix (e.g. NEB cat # M0287)
  • Magnet for 96 well plates (for 96 barcodes)
  • Multichannel pipette (for 96 barcodes)
  • 0.2 ml 96-well PCR plates and seals (for 96 barcodes)

Premium whole genome amplification

  • Qiagen REPLI-g Midi Kit
  • T7 Endonuclease I (NEB, cat # M0302)
  • TE buffer: 10 mM Tris (pH 8.0), 0.1 mM EDTA
  • PEG 8000, 50% w/v
  • 0.5 M EDTA, pH 8
  • 5 M NaCl (Sigma, cat # 71386)
  • 1 M Tris-HCl pH 8.0

Compatibility

The Ligation Sequencing Kit can be used together with:

Kits

  • PCR Barcoding Expansion 1-12 (EXP-PBC001)
  • PCR Barcoding Expansion 1-96 (EXP-PBC096)
  • Native Barcoding Expansion 1-12 (EXP-NBD104)
  • Native Barcoding Expansion 13-24 (EXP-NBD114)
  • Native Barcoding Expansion 96 (EXP-NBD196)
  • PCR Expansion (EXP-PCA001)
  • Flow Cell Wash Kit (EXP-WSH004)

Flow cells

  • FLO-MIN106D
  • FLO-PRO002
  • FLO-FLG001

Devices

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