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Oxford Nanopore TechnologiesFully scalable, real-time DNA/RNA sequencing technology
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London Calling 2022
18th - 20th May
Native Barcoding Kit 96 (Q20+)
SQK-NBD112.96

A versatile method of preparing barcoded sequencing libraries optimised for modal raw read accuracy of Q20+ (99%+) and long reads.

This is an Early Access product

This product has a 2 week lead time

$799.00
2Early Access
  • Access the latest products from Oxford Nanopore Technologies
  • Provide feedback into the innovation team as the product develops
  • Product Warranty may initially be limited to 1 month as it is so new
  • Product routinely iterates as new features and capabilities are added
  • Estimated lead time in store
  • Product subject to availability

Information

This kit is recommended for users who:

  • want to achieve raw read sequencing modal accuracy of Q20+ (99%+)
  • want to optimise their sequencing experiment for accuracy
  • wish to multiplex samples to reduce price per sample
  • need a PCR-free method of multiplexing to preserve additional information such as base modifications
  • require control over read length
  • are interested in utilising upstream processes such as size selection or whole genome amplification

This is an Early Access product
For more information about our Early Access programmes, please see this article on product release phases..

The Native Barcoding Kit 96 features:

FeatureProperty
Preparation time140 minutes
Input requirement400 ng per sample of gDNA
200 fmol (130 ng for 1 kb amplicons) per sample of amplicons
PCR RequiredNo
FragmentationOptional
Read lengthEqual to fragment length
Read type produced1D
Associated protocolsLigation sequencing gDNA - native barcoding (SQK-NBD112.96)
Native Barcoding Kit 96 with amplicons (SQK-NBD112.96)
Pack size12 reactions
Options include: 3 libraries containing 96 barcodes, 6 libraries containing 48 barcodes or 12 libraries containing 24 barcodes
StabilityShipped at 2–8°C

Long-term storage -20°C

Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer

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:

No Barcodes24 barcodes48 barcodes96 barcodes
Flow cell price$500$500$500$500
Library price$99$99$99$99
Barcode price-$75$150$300
Price per sample$599$28$15$9.36

The Native Barcoding Kit 96 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 ends are 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 adapters.

The kit is optimised to achieve sequencing accuracies of over 99% (Q20+). For highest data yields, we recommend starting with 400 ng per sample for both R9.4.1 and R10.4 flow cells. Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and can reduce sequencing output.

This Native Barcoding Kit (Kit 12) includes the Adapter Mix II H (AMII H), with three key features: Firstly, the adapter is loaded with an updated sequencing enzyme enabling accuracies of over 99% (Q20+). Secondly, the adapter is higher capture, enabling lower flow cell loading amounts. Finally, the adapter contains the fuel fix technology enabling users to run long 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. Please 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 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, using the BluePippin, for example). 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 throughput. 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; DNA ends are 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 adapters.

Native barcoding workflow1

Protocols

Ligation sequencing gDNA - native barcoding (SQK-NBD112.96)
Native Barcoding Kit 96 with amplicons (SQK-NBD112.96)

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

What's in the box

The Native Barcoding Kit 96 contains 96 unique barcodes and sufficient reagents for 12 reactions (options include: 3 libraries containing 96 barcodes, 6 libraries containing 48 barcodes, or 12 libraries containing 24 barcdoes)

SQK-NBD112.96

NameAcronymCap colourNo. of vialsFill volume per vial (µl)
Adapter Mix II HAMII HGreen240
Sequencing Buffer IISBIIRed2500
Loading Beads IILBIIPink2360
Loading SolutionLSWhite cap, pink label2400
Elution BufferEBBlack1500
AMPure XP BeadsAXPWhite16,000
L Fragment BufferLFBWhite17,500
S Fragment BufferSFBWhite17,500
EDTAEDTAClear1700
Flush BufferFBWhite115,500
Flush TetherFLTPurple1400

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.

3rd party materials

Consumables

  • NEB Blunt/TA Ligase Master Mix (M0367)
  • NEBNext® Quick Ligation Reaction Buffer (NEB B6058)
  • NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (cat # E7180S)
  • Alternatively to the NEBNext® Companion Module and the NEBNext® Quick Ligation Reaction Buffer, you can use the three NEBNext® products below:
  • NEBNext FFPE Repair Mix (M6630)
  • NEBNext Ultra II End repair/dA-tailing Module (E7546)
  • NEBNext Quick Ligation Module (E6056)
  • Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Cat # 0030129504) with heat seals
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • Freshly prepared 70% ethanol in nuclease-free water
  • Qubit™ Assay Tubes (ThermoFisher Q32856)
  • Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)

Equipment

  • Hula mixer (gentle rotator mixer)
  • Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, #11766427)
  • 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

Optional equipment

  • Agilent Bioanalyzer (or equivalent)
  • Qubit fluorometer (or equivalent for QC check)
  • Eppendorf 5424 centrifuge (or equivalent)

Compatibility

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

Kits

  • Sequencing Auxiliary Vials (EXP-AUX002)
  • Flow Cell Wash Kit (EXP-WSH004)
  • Native Barcoding Expansion (EXP-NBD112)

Flow cells

  • FLO-MIN106D
  • FLO-MIN112
  • FLO-PRO002
  • FLO-PRO112

Devices

  • MinION Mk1B
  • MinION Mk1C
  • GridION
  • PromethION