Information
This is an Early Access product.
This kit is recommended for users who:
- want to do full-length sequencing of 16S rRNA for bacterial profiling and ITS for fungal profiling
- are interested in genus-level bacterial identification
- are looking for a rapid workflow that is faster and more cost-effective than a ligation-based approach
- want a fragmentation-free workflow that preserves full-length amplicons
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.
*Multiplexing samples onto one flow cell can reduce the cost per sample for a user.
The Microbial Amplicon Barcoding Sequencing Kit 24 V14 offers a method of amplifying the entire 16S rRNA or ITS gene from extracted gDNA, followed by library preparation. This kit contains updated primers that enable full-length sequencing of 16S rRNA for bacterial profiling and ITS for fungal profiling from diverse sample types.
Please note: Our primer schemes are aligned with industry standard and provide coverage for the entire 16S rRNA and ITS rRNA gene respectively. However, the primers used bind within regions that are not 100% conserved across species and therefore we are unable to guarantee amplification for all species. Please ensure you run a control experiment to determine if your targeted species will be amplified using the primers provided in this kit. If you require further support contact us via support@nanoporetech.com.
The Microbial Amplicon Barcoding Sequencing Kit 24 V14 features:
| Feature | Property |
|---|
| Preparation time | 70 minutes + ~60 minutes PCR |
| Input requirement | 10 ng gDNA per sample |
| PCR required | Yes |
| Fragmentation | No |
| Read length | Full-length 16S (~1.5 kb) and/or ITS gene (variable) |
| Kit chemistry | Kit 14 |
| Associated protocols | Microbial Amplicon Barcoding Sequencing for 16S and ITS (SQK-MAB114.24) |
| Multiplexing options | • Microbial Amplicon Barcoding Sequencing Kit 24 V14
• Flow Cell Wash Kit |
| Pack size | 6 reactions |
| Stability | Shipped 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 |
The DNA is amplified by PCR using specific 16S or ITS primers. The amplicons are then barcoded. These barcodes contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
This kit uses our Kit 14 chemistry, which includes improved modal raw read sequencing accuracies with high output on our latest nanopore: R10.4.1. The flow cell priming and sequencing reagents are also included as part of the sequencing kit and are compatible with the R10.4.1 nanopore. This chemistry presents a higher capture rate of gDNA 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.
The kit is supported by the EPI2ME wf-16S workflow, which can be used to analyse data from the Microbial Amplicon Barcoding Sequencing Kit 24 V14 (SQK-MAB114.24) protocol. Deconvolution of barcoded sequencing data is supported by Dorado and EPI2ME which classify the barcode sequence and sort reads into corresponding folders.
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 DNA is amplified by PCR using specific 16S or ITS primers. Up to 24 amplicon samples are then barcoded. These barcodes contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.
The kit is supported by the EPI2ME wf-16S workflow, which can be used to analyse data from the Microbial Amplicon Barcoding Sequencing Kit 24 V14 (SQK-MAB114.24) protocol. Deconvolution of barcoded sequencing data is supported by Dorado and EPI2ME which classify the barcode sequence and sort reads into corresponding folders.
What's in the box
The Microbial Amplicon Barcoding Sequencing Kit 24 V14 contains 24 unique barcodes and sufficient reagents to generate six sequencing libraries:
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|
| Rapid Adapter | RA | Green | 1 | 15 |
| 16S Primers | 16S | White | 1 | 800 |
| ITS Primers | ITS | White | 1 | 800 |
| S Fragment Buffer | SFB | Clear cap, white label | 2 | 1,800 |
| EDTA | EDTA | Blue | 1 | 700 |
| Adapter Buffer | ADB | Clear | 1 | 100 |
| AMPure XP Beads | AXP | Brown | 2 | 1,200 |
| Elution Buffer | EB | Black | 1 | 500 |
| Sequencing Buffer | SB | Red | 1 | 700 |
| Library Beads | LIB | Pink | 1 | 600 |
| Library Solution | LIS | White cap, pink label | 1 | 600 |
| Flow Cell Flush | FCF | Clear cap, light blue label | 1 | 8,000 |
| Flush Tether UL | FTU | Purple | 1 | 65 |
| Amplicon Barcodes 01-24 | AB24 | - | 2 plates, 3 sets of barcodes per plate | 5 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.
3rd party materials
Consumables
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
- (Optional) Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Freshly prepared 80% ethanol in nuclease-free water
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment
- Ice bucket with ice
- Microfuge
- Timer
- Thermal cycler
- Magnetic rack
- Hula mixer (gentle rotator mixer)
- P1000 pipette with tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P2 pipette and tips
- Multichannel pipette
- Qubit fluorometer (or equivalent for QC check)
Note: The above are 3rd party reagents required for the standard prep. For other workflows using this kit, extra 3rd party reagents may be required.
