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Ligation Sequencing Kit XL

SQK-LSK109-XL

$4,000.00

A versatile sequencing kit optimised for throughput, long reads, and processing multiple samples simultaneously.

gDNA / amplicon / cDNA ≤ 1 µg No PCR Throughput
  • Information

    This kit is recommended for users who:

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

    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 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. This kit is a scaled-up version of the 6-reaction Ligation Sequencing Kit, and contains larger quantities of the same components. This allows users to prepare multiple libraries using a multichannel pipette or a liquid-handling robot.

    The kit is optimised to generate maximum throughput and read length. 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 throughput.

    Users can either start with 1 μg of gDNA, quantified using the Qubit fluorometer, 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, 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 Circulomics Short Read Eliminator Kit, for example). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.

    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 Sequencing 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 PCR-cDNA Sequencing Kit or the Direct cDNA Sequencing Kit.

    Shipping and logistics:

    Flow cells and kits are shipped together in a temperature-controlled shipping box with gel ice packs. Ice packs are stored at 2-8° C, and -20° C ice packs are added to the top of the box (but not in direct contact with product) to maintain the integrity of the shipment.

    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.

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

  • 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.

  • Protocols
  • Safety and legal
  • What's in the box

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

    LSK109 Contents

    The Flow Cell Priming Kit XL is also supplied.

    EXP-FLP002-XL

  • 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
    • 15 or 50 ml Falcon tubes

    Optional consumables

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

    Equipment

    • Hula mixer (gentle rotator mixer)
    • Magnetic separator:
      •  Either suitable for 0.2 ml PCR tubes, e.g. DynaMag™-PCR Magnet (Thermo Fisher, #492025) or DynaMag™-96 Bottom Magnet (Thermo Fisher, #12332D)
      • OR suitable for 96-well plates, e.g. Magnum FLX® Enhanced Universal Magnet Plate (Alpaqua)
      • Microfuge
      • Vortex mixer
      • Thermal cycler
      • Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl
      • 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)
  • Compatibility

    The Ligation Sequencing Kit XL can be used together with:

    Kits

    • PCR Expansion (EXP-PCA001)
    • Flow Cell Wash Kit (EXP-WSH003)

    Flow cells

    • FLO-MINSP6
    • FLO-MIN106D
    • FLO-MIN111
    • FLO-PRO002
    • FLO-FLG001

    Devices

    • MinION Mk1B
    • MinION Mk1C
    • Flongle
    • GridION Mk1
    • PromethION P24/P48
Information

This kit is recommended for users who:

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

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 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. This kit is a scaled-up version of the 6-reaction Ligation Sequencing Kit, and contains larger quantities of the same components. This allows users to prepare multiple libraries using a multichannel pipette or a liquid-handling robot.

The kit is optimised to generate maximum throughput and read length. 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 throughput.

Users can either start with 1 μg of gDNA, quantified using the Qubit fluorometer, 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, 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 Circulomics Short Read Eliminator Kit, for example). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.

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 Sequencing 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 PCR-cDNA Sequencing Kit or the Direct cDNA Sequencing Kit.

Shipping and logistics:

Flow cells and kits are shipped together in a temperature-controlled shipping box with gel ice packs. Ice packs are stored at 2-8° C, and -20° C ice packs are added to the top of the box (but not in direct contact with product) to maintain the integrity of the shipment.

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.

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

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.

What's in the box

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

LSK109 Contents

The Flow Cell Priming Kit XL is also supplied.

EXP-FLP002-XL

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
  • 15 or 50 ml Falcon tubes

Optional consumables

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

Equipment

  • Hula mixer (gentle rotator mixer)
  • Magnetic separator:
    •  Either suitable for 0.2 ml PCR tubes, e.g. DynaMag™-PCR Magnet (Thermo Fisher, #492025) or DynaMag™-96 Bottom Magnet (Thermo Fisher, #12332D)
    • OR suitable for 96-well plates, e.g. Magnum FLX® Enhanced Universal Magnet Plate (Alpaqua)
    • Microfuge
    • Vortex mixer
    • Thermal cycler
    • Multichannel pipettes suitable for dispensing 2–20 μl and 20–200 μl
    • 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)
Compatibility

The Ligation Sequencing Kit XL can be used together with:

Kits

  • PCR Expansion (EXP-PCA001)
  • Flow Cell Wash Kit (EXP-WSH003)

Flow cells

  • FLO-MINSP6
  • FLO-MIN106D
  • FLO-MIN111
  • FLO-PRO002
  • FLO-FLG001

Devices

  • MinION Mk1B
  • MinION Mk1C
  • Flongle
  • GridION Mk1
  • PromethION P24/P48
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