This kit is recommended for users who:

• Want to optimise their sequencing experiment for throughput
• Want to use upstream processes such as size selection, whole-genome amplification, or enrichment for long reads

This protocol describes how to carry out sequencing of a DNA sample using the Q-Line Ligation Sequencing Kit (Q-SQK-LSK114). This kit is designed to prepare genomic DNA, amplicons, and cDNA, with or without barcoding, for sequencing on Oxford Nanopore devices to produce 1D reads. The kit provides enough material for six reactions. We recommend that new users test this protocol using the Lambda Control Expansion Kit (Q-EXP-CTL001) as an input.

The library preparation involves two enzymatic steps to prepare the DNA ends for sequencing. The first enzymatic step repairs any damage in the DNA molecules, such as nicks, and generates uniform ends with 5’ phosphates and 3’ adenine overhangs. The second enzymatic step ligates the sequencing adapters that have complementary thymine tails with the dA-tailed template.

Prepare for your experiment

You will need to:

• Extract your DNA and check its length, quantity, and purity. The quality checks performed during the protocol are essential to ensure experimental success.
• Ensure you have your sequencing kit, the correct equipment, and third-party reagents.

Library preparation

You will need to:

• Repair nicks in the DNA and prepare the DNA ends for adapter attachment.
• Attach sequencing adapters, supplied in the kit, to the DNA ends.
• Prime the flow cell and load your DNA library into the flow cell.

Sequencing and analysis

You will need to start an assay using the sequencing software, which will collect raw data from the device and convert it into basecalled reads.

Warnings

• Follow Good Laboratory Practice in relation to personal safety, including but not limited to the use of personal protective equipment when handling the samples and test reagents. Refer to Safety Data Sheets provided with the product for specific safety requirements.
• Take care to prevent the reagents from contacting your skin, eyes, and mucous membranes.
• If contact occurs, immediately wash the affected area with soap and water. Thoroughly wash hands after handling samples and test reagents.
• In the event of a serious incident, contact the Manufacturer of the device. The user may also wish to contact the competent authority of the Member State in which the device was used.
• The Q-Line GridION as supplied provides only raw sequence data, which requires validated downstream analysis and the inclusion of assay-specific controls.
• Using the Q-Line GridION outside of the intended use is not recommended and is at the user’s risk.

Precautions

• Refer to local or WHO health and safety guidance for handling samples containing hazard group (HG)/BioSafety Level (BSL) 3 organisms.
• Decontaminate surfaces with 0.5% sodium hypochlorite (bleach) diluted with deionised or purified water.
• Prevent contamination of reagents by following standard contamination prevention procedures including but not limited to the use of personal protective equipment and using filter pipette tips for handling DNA.
• Reagents must be stored under the indicated storage conditions before and after use.
• Waste should be disposed of in accordance with local waste disposal regulations.
• Do not mix different lots of reagents.
• We recommend using DNase-free pipette tips.
• Never reuse the consumables intended for single use (for example, tips, gloves, and test tubes).
• When using a pipette, use correct volumes and adhere to manufacturer’s instructions. The use of multichannel pipettes is recommended where possible. Mouth pipettes must not be used.
• Do not use the reagents if the package is damaged or reagent tubes are leaking (reagents may be compromised, leading to an incorrect result). Please contact technical support.
• Do not use test reagents after their expiration date.
• Do not use the flow cell if the package is damaged or the flow cell is leaking as the flow cell may be compromised, leading to an incorrect result. If leakage occurs, refer to the Flow Cell Safety Data Sheet for handling guidance, and contact technical support.
• Following recalibration, flow cells should be used within 7 days.
• After use, the flow cell must be prepared for return and disposal as described in the Q-Line GridION user guide.

Library description	Sequencing run time	Accuracy	Data output
48 kb fragment	24 hours	~99.3% modal single-read accuracy	~9.5 Gb
1 kb amplicon	24 hours	~99.4% modal single-read accuracy	~12.5 Gb

Statistics are representative of average observed performance in verification tests. Values vary depending on sample quality, library preparation method, flow cell type, and basecalling model. Values provided assume high-quality samples that are prepared following our optimised protocols.

How to QC your input DNA

It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.

Chemical contaminants

Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

Name	Abbreviation	Cap colour	Quantity	Minimum fill volume per container (µl)
Flow Cell Flush	FCF	N/A	1	7,020
Flow Cell Tether	FCT	Purple	1	180
Elution Buffer	EB	Black	1	90
S Fragment Buffer	SFB	Clear	2	1,500
L Fragment Buffer	LFB	Orange	2	1,500
Library Beads	LIB	Pink	1	153
Ligation Adapter	LA	Green	1	30
Sequencing Buffer	SB	Red	1	225
AMPure XP Beads	AXP	Amber	1	600
Ligation Buffer	LNB	White	1	150

Note: The AMPure XP reagent manufactured by Beckman Coulter, Inc. can be stored at –20°C with the kit without detriment to reagent stability.

The Q-Line GridION device contains all the hardware required to control up to five sequencing experiments simultaneously and acquire the data. The device is further enhanced with high-performance GPU technology for real-time basecalling. Read more in the Q-Line GridION configuration guide.

The Sequencing Software controls the GridION, collects sequencing data in real-time and processes it into basecalled reads. The software can also demultiplex reads by barcode, and basecall/demultiplex data after a sequencing run has completed. You will be using the Sequencing Software for every assay you run.

For instructions on how to run the Sequencing Software on the GridION, please refer to the Q-Line GridION user guide.

Note: This protocol describes the process of starting a sequencing run using the default Q-SQK-LSK114 assay supplied with Q-Line GridION. For more information on setting up a sequencing run, see the Q-Line GridION user guide. For more information on configuring an assay definition file for your own assay, see the Q-Line GridION configuration guide. Ensure you select the correct assay for your experiment.