This kit is recommended for users who:

• Want to multiplex samples to reduce price per sample
• Need a PCR-free method of multiplexing to preserve additional information such as base modifications
• Require a short preparation time
• Have limited access to laboratory equipment

This protocol describes how to carry out rapid barcoding of genomic, plasmid, or amplicon DNA using the Q-Line Rapid Barcoding Kit 96 V14 (Q-SQK-RBK114.96). There are 96 unique barcodes, allowing the user to pool up to 96 different samples in one sequencing experiment.

The DNA input undergoes a transposase step to fragment the samples while attaching rapid barcodes to the ends simultaneously. The samples are then pooled and a bead clean-up performed to remove any excess transposases in the reaction. The sequencing adapters are then attached to the barcodes and the library can be sequenced.

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 the Q-Line Rapid Barcoding Kit 96 V14, the correct equipment, and third-party reagents.

Library preparation

You will need to:

• Tagment your DNA using the Rapid Barcodes in the kit; this simultaneously attaches a pair of barcodes to the fragments.
• Pool the barcoded samples.
• 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 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 - 96 samples	24 hours	~99.3% modal single-read accuracy	~8.1 Gb / >50 Mb per sample
1 kb amplicon - 24 samples	24 hours	~99.4% modal single-read accuracy	>50 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.

インプットDNAのQC方法

インプットDNAの量と品質の要件を満たすことが重要です。DNAの使用量が少なすぎたり多すぎたり、あるいは品質の低いDNA（例としてDNAが非常に断片化されていたり、RNAや化学汚染物質が含まれている場合など）を使用すると、ライブラリーの調製に影響を及ぼす可能性があります。

DNAサンプルの品質管理の方法については、Input DNA/RNA QC protocolのプロトコルをご覧ください。

コンタミネーション

DNAの抽出する方法によっては、精製DNAに特定の化学汚染物質が残留する可能性があり、ライブラリ調製の効率やシークエンシングの品質に影響を及ぼす可能性があります。コンタミネーションについての詳細は、コミュニティーの Contaminants page をご覧ください。

Name	Abbreviation	Cap colour	Quantity	Minimum fill volume per container (µl)
AMPure XP Beads	AXP	Amber	3	1,000
Elution Buffer	EB	Black	1	720
Rapid Adapter	RA	Green	2	9
Adapter Buffer	ADB	Clear	1	42
Library Beads	LIB	Pink	1	306
Sequencing Buffer	SB	Red	1	450
Flow Cell Flush	FCF	N/A	1	14,040
Flow Cell Tether	FCT	Purple	2	180
96 Rapid Barcodes	RB01–RB96	N/A	3	1.5 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.

Note: This protocol describes the process of starting a sequencing run using the default Q-SQK-RBK114.96 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.