Information

This kit is recommended for users who:

• Wish to multiplex up to 96 samples to reduce price per sample
• Want to achieve modal raw read accuracy of Q20+ (99%) and above
• Want to optimise their sequencing experiment for accuracy and output
• Need a PCR-free method of multiplexing to preserve additional information such as base modifications
• Require control over read length
• Would like to utilise upstream processes such as size selection or whole genome amplification

Please note that to use this kit, you will need to purchase additional 3rd party reagents: see the "3rd Party Materials" tab for more detail.

Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow cell: it allows a user to pool multiple samples and sequence them together, making more efficient use of the flow cell.

Multiplexing samples onto one flow cell can reduce the cost per sample for a user. Below are some worked examples. Please note, this is an example and pricing depends on the kit purchased.

	No barcodes	24 barcodes	48 barcodes	96 barcodes
Flow cell price	$500	$500	$500	$500
Library price	$99	$99	$99	$99
Barcodes price	-	$75	$150	$300
Price per sample	$599	$28	$15	$9.36

The Native Barcoding Kit 96 V14 features:

Feature	Property
Preparation time	140 minutes
Input requirement	• 400 ng per sample of gDNA • 200 fmol (130 ng for 1 kb amplicons) per sample of amplicons
PCR Required	No
Fragmentation	Optional
Kit chemistry	Kit 14 (V14)
Read length	Equal to fragment length
Associated protocols	• Ligation Sequencing gDNA - Native barcoding kit 96 V14 • Ligation Sequencing amplicons - Native barcoding kit 96 V14 • Ligation sequencing influenza whole genome V14 (SQK-NBD114.24 or SQK-NBD114.96) • Coming soon: Classic PCR tiling of SARS-CoV-2 virus with the Native Barcoding Kit 96 (SQK-NBD114.96)
Pack size	Up to 12 reactions: Options: 3 libraries containing 96 barcodes, 6 libraries containing 48 barcodes or 12 libraries containing 24 barcodes
Stability	Shipped at 2–8°C Long-term storage -20°C The warranty for this Early Access product is 3 months from receipt by the customer

The Native Barcoding Kit 96 V14 is a standalone kit providing 96 unique barcodes to enable PCR-free multiplexing of dsDNA samples such as gDNA and amplicons.

The library preparation method is similar to the Ligation Sequencing Kit protocol; gDNA is repaired (note: DNA repair is skipped when using amplicon input) and dA-tailed using the NEBNext End Repair/dA-tailing module, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end and this is used as a hook to ligate to the supplied sequencing adapter.

The kit is optimised to achieve modal sequencing accuracies of over 99% (Q20+) with high output on our latest nanopore: R10.4.1. Our flow cell priming and sequencing reagents have been reformulated to be compatible with this improved Kit 14 adapter and R10.4.1 nanopore.

Kit 14 also includes previous updates such as the higher capture rate of DNA to enable lower flow cell loading amounts, and fuel fix technology, allowing users to run longer experiments without the need for fuel addition during the run.

If your experiment requires long reads, it is recommended to start with full-length gDNA, and fragmentation/shearing is neither advised nor required. Note: If long reads are required, high molecular weight DNA should be used as input. To determine purity, we suggest using the Nanodrop to measure the A260/280 and A260/230 ratios and we recommend that the sample should meet the following criteria:

• A260/280 = 1.8
• A260/230 = 2.0-2.2

The Native Barcoding Kit 96 V14 is compatible with upstream processes such as whole genome amplification (for applications where under 1 ng of sample is available) and size selection (for enrichment of specified fragment lengths, for example, using our Short Fragment Eliminator Kit (EXP-SFE001)). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.

Deconvolution of barcoded sequencing data is supported by MinKNOW, Guppy and EPI2ME which classify the barcode sequence and sort reads into corresponding folders.

Further considerations:

Starting with lower amounts of input material, or impure samples can affect library preparation efficiency and lower sequencing output. PCR can be used to generate more input material in cases where sample amount is limiting.

Shipping and logistics:

Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.

Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.

The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.

Workflow

The library preparation method is similar to the Ligation Sequencing Kit protocol; gDNA is FFPE repaired (note: DNA repair is skipped when using amplicon input) and end-prepped/dA-tailed using the NEBNext End Repair/dA-tailing module. A unique dT-tailed barcode adapter is then ligated on the dA-tailed template. Barcoded samples are pooled together. Each barcode adapter also has a cohesive end which is used as a hook to ligate to the supplied sequencing adapter.

Protocols

• Ligation sequencing gDNA - native barcoding kit 96 V14
• Ligation sequencing amplicon - native barcoding kit 96 V14
• Ligation sequencing influenza whole genome V14 (SQK-NBD114.24 or SQK-NBD114.96)

To access the protocol, you will need to register for a Nanopore Community account.

Safety and legal

Safety and Data Sheet for Native Barcoding Kit 96 V14 (SQK-NBD114.96) with higher concentration EDTA

Safety Data Sheet for Native Barcoding Kit 96 V14 (SQK-NBD114.96) with reduced concentration EDTA

What's in the box

The Native Barcoding Kit 96 V14 contains 96 unique barcodes and sufficient reagents for up to 12 reactions.

Note: Our kits have been updated with reduced EDTA concentration.

Reduced EDTA concentration format (current):

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Native Barcode plate	NB01-96	-	3 plates	8 µl per well
DNA Control Sample	DCS	Yellow	3	35
Native Adapter	NA	Green	2	40
Sequencing Buffer	SB	Red	2	700
Library Beads	LIB	Pink	2	600
Library Solution	LIS	White cap, pink label	2	600
Elution Buffer	EB	Black	1	1,500
AMPure XP Beads	AXP	Clear cap, light teal	1	6,000
Long Fragment Buffer	LFB	Clear cap, orange label	1	7,500
Short Fragment Buffer	SFB	Brown cap, dark blue label	1	7,500
EDTA‡	EDTA	Blue	1	700
Flow Cell Flush	FCF	Clear cap, light blue label	1	15,500
Flow Cell Tether	FCT	Purple	2	200

‡ Reduced concentration of EDTA with a blue cap.

Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

Higher EDTA concentration format (old):

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Native Barcode plate	NB01-96	-	3 plates	8 µl per well
DNA Control Sample	DCS	Yellow	3	35
Native Adapter	NA	Green	2	40
Sequencing Buffer	SB	Red	2	700
Library Beads	LIB	Pink	2	600
Library Solution	LIS	White cap, pink label	2	600
Elution Buffer	EB	Black	1	1,500
AMPure XP Beads	AXP	Clear cap, light teal label	1	6,000
Long Fragment Buffer	LFB	Clear cap, orange label	1	7,500
Short Fragment Buffer	SFB	Brown cap, dark blue label	1	7,500
EDTA†	EDTA	Clear	1	700
Flow Cell Flush	FCF	Clear cap, light blue label	1	15,500
Flow Cell Tether	FCT	Purple	2	200

† Higher concentration of EDTA with a clear cap.

3rd party materials

Consumables

• NEB Blunt/TA Ligase Master Mix (M0367)
• NEBNext FFPE Repair Mix (M6630)
• NEBNext Ultra II End repair/dA-tailing Module (E7546)
• NEBNext Quick Ligation Module (E6056)
• 2 ml Eppendorf DNA LoBind tubes
• 1.5 ml Eppendorf DNA LoBind tubes
• 0.2 ml thin-walled PCR tubes or PCR plate
• Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
• Freshly prepared 80% ethanol in nuclease-free water
• Qubit™ Assay Tubes (ThermoFisher Q32856)
• Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)
• (Optional) Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, cat# AM2616)

Equipment

• Hula mixer (gentle rotator mixer)
• Microfuge
• Magnetic rack
• Vortex mixer
• Thermal cycler
• Multichannel pipette
• P1000 pipette and tips
• P200 pipette and tips
• P100 pipette and tips
• P20 pipette and tips
• P10 pipette and tips
• P2 pipette and tips
• Ice bucket with ice
• Timer
• Qubit fluorometer (or equivalent for QC check)

Optional equipment

• Agilent Bioanalyzer (or equivalent)
• Eppendorf 5424 centrifuge (or equivalent)

Compatibility

The Native Barcoding Kit 96 V14 can be used together with:

Kits

• Native Barcoding Auxiliary Kit V14 (EXP-NBA114)
• Flow Cell Wash Kit (EXP-WSH004)
• Flow Cell Priming Kit (EXP-FLP004)
• Flow Cell Priming Kit XL (EXP-FLP004-XL)
• Sequencing Auxiliary Vials V14 (EXP-AUX003)
• LFB Expansion (EXP-LFB001)
• SFB Expansion (EXP-SFB001)

Flow cells

R10.4.1.

• FLO-MIN114
• FLO-PRO114M
• FLO-FLG114

Devices

• Flongle
• MinION Mk1B
• MinION Mk1C
• GridION
• PromethION