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Ligation Sequencing Kit

SQK-LSK109

$599.00

A ligation-based sequencing kit for multiplexing samples.  
For singleplex samples, please use our new version: SQK-LSK110.
SQK-LSK109 is to be discontinued on May 10, 2021.

Includes a Flow Cell Priming Kit

gDNA / amplicon / cDNA ≤ 1 µg 60 mins No PCR Throughput
  • 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.

    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:

    Feature Property
    Preparation time 60 minutes
    Input requirement 1000 ng of double-stranded DNA
    PCR Required No
    Fragmentation Optional; recommended for inputs of 100-500 ng
    Read length Equal to fragment length
    Read type produced 1D
    Typical throughput* •••
    Associated protocols

    Ligation Sequencing Kit

    Premium whole genome amplification

    Sequence capture

    Long read selection by Blue Pippin

    Multiplexing options

    Native Barcoding Expansion 1-12 and 13-24

    PCR Barcoding Expansion 1-12 and 1-96

    Flow Cell Wash Kit

    Pack size 6 reactions
    Stability

    Shipped at  2–8° C

    Long-term storage  -20° C

    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.

    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.

    Compare Sequencing Kits

    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 six sequencing libraries.

     

    The Flow Cell Priming Expansion Pack is also supplied.

    EXP-FLP002

  • Multiplexing

    PCR-free multiplexing with barcoding for up to 24 samples is available with additional purchases: Native Barcoding Expansion 1-12 (EXP-NBD104) and Native Barcoding Expansion 13-24 (EXP-NBD114).

    PCR-based multiplexing with barcoding for up to 12 samples is available with an alternative purchase: PCR Barcoding Kit (SQK-PBK004), or Rapid PCR Barcoding Kit (SQK-RPB004).

    Multiplexing with barcoding for up to 96 samples is available (this option requires PCR) with an additional purchase: PCR Barcoding Expansion 1-12 (EXP-PBC001), or PCR Barcoding Expansion 1-96 (EXP-PBC096).

  • 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:

    1D sequencing selecting for long reads

    • BluePippin device and gel cassette

    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)
    • Multi-channel 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)
    • 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:

  • 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 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:

Feature Property
Preparation time 60 minutes
Input requirement 1000 ng of double-stranded DNA
PCR Required No
Fragmentation Optional; recommended for inputs of 100-500 ng
Read length Equal to fragment length
Read type produced 1D
Typical throughput* •••
Associated protocols

Ligation Sequencing Kit

Premium whole genome amplification

Sequence capture

Long read selection by Blue Pippin

Multiplexing options

Native Barcoding Expansion 1-12 and 13-24

PCR Barcoding Expansion 1-12 and 1-96

Flow Cell Wash Kit

Pack size 6 reactions
Stability

Shipped at  2–8° C

Long-term storage  -20° C

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.

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.

Compare Sequencing Kits

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
What's in the box

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

 

The Flow Cell Priming Expansion Pack is also supplied.

EXP-FLP002

Multiplexing

PCR-free multiplexing with barcoding for up to 24 samples is available with additional purchases: Native Barcoding Expansion 1-12 (EXP-NBD104) and Native Barcoding Expansion 13-24 (EXP-NBD114).

PCR-based multiplexing with barcoding for up to 12 samples is available with an alternative purchase: PCR Barcoding Kit (SQK-PBK004), or Rapid PCR Barcoding Kit (SQK-RPB004).

Multiplexing with barcoding for up to 96 samples is available (this option requires PCR) with an additional purchase: PCR Barcoding Expansion 1-12 (EXP-PBC001), or PCR Barcoding Expansion 1-96 (EXP-PBC096).

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:

1D sequencing selecting for long reads

  • BluePippin device and gel cassette

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)
  • Multi-channel 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)
  • 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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