Information

Ligation Sequencing Kit

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 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.
The kit contains an updated sequencing adapter (Adapter Mix F, or AMX-F), which is designed to turn over significantly less fuel during a sequencing run compared to previous kit versions. This results in better maintenance of enzyme speed and higher sequencing output.

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

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

Example protocol for sequencing gDNA by Ligation (SQK-LSK110) - for illustration purposes only

Safety and legal

Safety Data Sheet for Ligation Sequencing Kit (SQK-LSK110)

What's in the box

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

3rd party materials

Consumables

• Agencourt AMPure XP beads
• 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

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

Optional Equipment

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

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)
• PCR Expansion (EXP-PCA001)
• Sequencing Auxiliary Vials (EXP-AUX110)
• Flow Cell Wash Kit (EXP-WSH004)
• Native Barcoding kit upgrades coming soon

Flow cells

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

Devices

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