This kit is recommended for users who:

• Want to multiplex samples to reduce price per sample
• Have a low starting amount of DNA
• Require a simple library preparation procedure

This protocol describes how to carry out rapid low-input barcoding of genomic DNA using the Q-Line Rapid PCR Barcoding Kit 24 V14 (Q-SQK-RPB114.24). There are 24 unique barcodes, allowing the user to pool up to 24 different samples in one sequencing experiment.

The gDNA is prepared for sequencing by undergoing tagmentation to fragment the DNA and to simultaneously attach PCR primers to the fragment ends. PCR is performed and rapid barcode primers are attached to the DNA ends in the same step. After barcoding, the samples are pooled and the rapid sequencing adapter is attached to the ends of the DNA samples for sequencing.

Prepare for your experiment

You will need to:

• Extract your DNA and check its length, quantity and purity. The quality checks performed on input samples are essential to ensure experimental success.
• Ensure you have your sequencing kit, the correct equipment, and third-party reagents.
• Ensure a compatible assay is installed on your Sequencing Software.

Library preparation

You will need to:

• Tagment your DNA using the Fragmentation Mix in the kit.
• Perform PCR using the barcoded primer supplied in the kit.
• 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

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. There is also an option to demultiplex reads into barcode-specific folders.

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 barcodes	24 hours	~99.2% modal single-read accuracy	>400 Mb per sample
3.6 kb fragment - 24 barcodes	24 hours	~99.2% modal single-read accuracy	>400 Mb per sample
Microbial community - 24 barcodes	24 hours	~99.3% modal single-read accuracy	>450 Mb per sample

Statistics are representative of average observed performance in verification tests. Output per sample represents the minimum average output. 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.

DNA质控

选择符合质量和浓度要求的起始DNA至关重要的。使用过少或过多的DNA，或者质量较差的DNA（如，高度碎片化、含有RNA或化学污染物的DNA）都会影响文库制备。

有关如何对DNA样品进行质控，请参考起始DNA/RNA质控实验指南 。

化学污染物

从原始样本中提取DNA的方法不同，可能会导致经纯化的DNA中所残留的化学污染物不同。这会影响文库的制备效率和测序质量。请在牛津纳米孔社区的 Contaminants（污染物）页面 了解更多信息。

Name	Abbreviation	Cap colour	Quantity	Minimum fill volume per container (µl)
Fragmentation Mix	FRM	Amber	1	144
Rapid Adapter	RA	Green	1	9
Adapter Buffer	ADB	Clear	1	21
AMPure XP Beads	AXP	N/A	1	4,580
Elution Buffer	EB	Black	1	90
EDTA	EDTA	Blue	1	576
Sequencing Buffer	SB	Red	1	225
Library Beads	LIB	Pink	1	153
Flow Cell Flush	FCF	N/A	1	7,020
Flow Cell Tether	FCT	Purple	1	180
Rapid Barcode Primer 01–24	RLB01–24	N/A	2 plates, 3 sets of primer barcodes per plate	10 per well

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.

Samples will be diluted to the appropriate concentration and tagmented with PCR primer sites.

Samples will be barcoded with PCR primers, amplified and equimolar pools will be prepared for final libraries.