Information
This kit is recommended for users who:
- Wish to multiplex up to 24 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 | 6 barcodes | 12 barcodes | 24 barcodes |
|---|
| Flow cell price | $500 | $500 | $500 | $500 |
| Library price | $99 | $99 | $99 | $99 |
| Barcodes price | - | $25 | $50 | $75 |
| Price per sample | $599 | $104 | $54 | $28 |
The Native Barcoding Kit 24 V14 features:
The Native Barcoding Kit 24 V14 is a standalone kit providing 24 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 24 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 then pooled together. Each barcode adapter also has a cohesive end which is used as a hook to ligate to the supplied sequencing adapter.
What's in the box
The Native Barcoding Kit 24 V14 contains 24 unique barcodes and sufficient reagents for 6 reactions.
Note: We are in the process of reformatting the barcodes provided in this kit into a plate format. This will reduce plastic waste and will facilitate automated applications.
Plate format
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
|---|
| DNA Control Sample | DCS | Yellow | 2 | 35 |
| Native Adapter | NA | Green | 1 | 40 |
| Sequencing Buffer | SB | Red | 1 | 700 |
| Library Beads | LIB | Pink | 1 | 600 |
| Library Solution | LIS | White cap, pink label | 1 | 600 |
| Elution Buffer | EB | Black | 2 | 500 |
| AMPure XP Beads | AXP | Clear cap, light teal label | 1 | 6,000 |
| Long Fragment Buffer | LFB | Orange | 1 | 1,800 |
| Short Fragment Buffer | SFB | Clear | 1 | 1,800 |
| EDTA | EDTA | Blue | 1 | 700 |
| Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |
| Flow Cell Tether | FCT | Purple | 1 | 200 |
| Native Barcode plate | NB01-24 | - | 2 plates, 3 sets of barcodes per plate | 15 µl per well |
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.
Vial format
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
|---|
| Native Barcodes | NB01-24 | Clear | 24 (one per barcode) | 20 |
| DNA Control Sample | DCS | Yellow | 2 | 35 |
| Native Adapter | NA | Green | 1 | 40 |
| Sequencing Buffer | SB | Red | 1 | 700 |
| Library Beads | LIB | Pink | 1 | 600 |
| Library Solution | LIS | White cap, pink label | 1 | 600 |
| Elution Buffer | EB | Black | 2 | 500 |
| AMPure XP Beads | AXP | Clear cap, light teal label | 1 | 6,000 |
| Long Fragment Buffer | LFB | Orange | 1 | 1,800 |
| Short Fragment Buffer | SFB | Clear | 1 | 1,800 |
| EDTA | EDTA | Blue | 1 | 700 |
| Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |
| Flow Cell Tether | FCT | Purple | 1 | 200 |
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.
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)
Note: For use with the Ligation sequencing DNA V14 - dual barcoding (SQK-NBD114.24 with EXP-PBC096) protocol, extra reagents will be required.
Compatibility
The Native Barcoding Kit 24 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)
- PCR Barcoding Expansion 1-96 (EXP-PBC096)
- 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