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
- 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 features:
| Feature | Property |
|---|
| Preparation time | ~60 minutes |
| Input requirement | 1 µg of double-stranded gDNA or amplicons |
| PCR Required | No |
| Fragmentation | Optional; recommended for inputs of 100-500 ng |
| Read length | Equal to fragment length |
| Read type produced | 1D |
| Associated protocols | • Genomic DNA by Ligation
• Lambda Control Experiment
• Amplicons 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 size | Six reactions |
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 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 (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.
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 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 six sequencing libraries.
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
|---|
| DNA CS | DCS | Yellow | 1 | 35 |
| Adapter Mix H | AMX H | Green | 2 | 40 |
| AMPure XP Beads | AXP | Amber | 1 | 1,200 |
| Ligation Buffer | LNB | Clear | 1 | 200 |
| L Fragment Buffer | LFB | Orange | 2 | 1,800 |
| S Fragment Buffer | SFB | Clear | 2 | 1,800 |
| Sequencing Buffer II | SBII | Red | 1 | 500 |
| Elution Buffer | EB | Black | 1 | 1,200 |
| Loading Beads II | LBII | Pink | 1 | 360 |
| Loading Solution | LS | White cap, pink sticker on label | 1 | 400 |
| Flush Buffer | FB | Blue | 6 | 1,170 |
| Flush Tether | FLT | Purple | 1 | 200 |
Compatibility
The Ligation Sequencing Kit 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
