Ligation sequencing DNA V14 (SQK-LSK114)
Overview
The Ligation Sequencing Kit V14 (SQK-LSK114) protocol describes how to prepare a DNA sample for sequencing on a PromethION 24 instrument. It is designed for high-molecular-weight genomic DNA and does not require PCR. A Lambda control experiment is recommended for new users. Key steps include DNA extraction and quality control, DNA repair and end-prep, ligation of sequencing adapters and loading the library onto the flow cell.
Highlights
- PCR-free ligation-based library preparation suitable for high-molecular-weight genomic DNA and amplicons.
- Uses the Ligation Sequencing Kit V14 (SQK-LSK114); optional fragmentation and size selection can be applied to adjust library length.
- Library preparation comprises DNA repair and end-prep (~35 min), adapter ligation and clean-up (~20 min) and flow-cell priming/loading (10 min).
- Allows storage of the prepared library at 4 °C for short-term use or –80 °C for long-term storage.
- Data acquisition uses MinKNOW for basecalling; EPI2ME workflows can be used for downstream analysis.
Sample submission requirements
| Service | Sample type | Minimum amount | Quality | Fragmentation size |
|---|---|---|---|---|
| Ligation sequencing |
Whole |
≥3 µg DNA 30 ng/µL in 100 µL |
OD 260/280 = 1.8–2.0 260/230 = 2.0–2.2 |
Range from 2 kb to 13 kb |
- Please provide samples in nuclease-free water. If contamination is detected during QC using Bioanalyzer 2100, the cost of cleanup will be charged.
- Concentration must be measured by Qubit.
- The core has the right to determine whether submitted samples are qualified for the experiment.
- Please check the core ([email protected] or 713-500-7933) for more information.
Service workflow:
Data analysis
After adapter ligation, prime the flow cell and load the library. Start a sequencing run in MinKNOW, which collects and basecalls reads. Use EPI2ME or custom bioinformatic workflows to align reads and perform variant calling, assembly or other analyses appropriate to genomic DNA sequencing.
Useful documents
- Original protocol: Ligation sequencing DNA V14 (SQK?LSK114 (PDF).