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Ligation Sequencing Kit XL V14 features
This kit is recommended for users who:
- Would like to process multiple samples simultaneously, either with a multichannel pipette or a liquid-handling robot
- Would like to achieve raw read sequencing modal accuracy of Q20+ (99%) or above
- Require control over read length
- Would like to utilise upstream processes such as size selection, whole genome amplification, or enrichment for long reads
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Introduction to the automated Ligation Sequencing protocol for DNA
This protocol describes how to carry out sequencing of a DNA sample using the Ligation Sequencing Kit XL (SQK-LSK114-XL).
We have developed this automated protocol on the Tecan® DreamPrep® NGS liquid handling robot. This library preparation protocol is fully automated, except for the sample quantification steps and loading of the system.
Please note that this method is intended for research use only.
It is highly recommended that a Lambda control experiment is completed first to become familiar with the technology.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
Before starting - Manual steps:
- Extract your DNA and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
- Ensure you have your sequencing kit, the correct equipment, primed liquid-handling robot and third-party reagents
- Download the software for acquiring and analysing your data
- Check your flow cells to ensure they have enough pores for a good sequencing run
Prepare your library
You will need to:
Automated steps:
- Repair the DNA and prepare the DNA ends for adapter attachment
- Attach sequencing adapters supplied in the kit to the DNA ends
Manual steps:
- Quantify your DNA library as a quality control
- Prime the flow cell and load your DNA library into the flow cell
Overview of library preparation workflow:
Note: Timings are dependent on number of samples and include hands on time, such as deck loading and sample quantification
Sequencing and analysis
You will need to:
- Start a sequencing run using the MinKNOW software which will collect raw data from the device and convert it into basecalled reads
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Timings
Note: Timings are approximate and subject to change with updates.
Process X8 samples X24 samples X48 samples X96 samples Hands-on time Deck set-up and master mix preparation ~30 minutes Automated DNA repair, end-prep and clean-up 1 hour 1 hour 1 hour 5 minutes 1 hour 10 minutes Automated adapter ligation and clean-up 1 hour 30 minutes 1 hour 30 minutes 1 hour 35 minutes 1 hour 35 minutes Quantification ~10 minutes Total 2 hours 30 minutes 2 hours 30 minutes 2 hours 40 minutes 2 hours 45 minutes ~40 minutes Sequencing run set-up and flow cell loading timings are variable depending on the number of samples and user experience.