Microbial Amplicon Barcoding Sequencing for 16S and ITS (SQK-MAB114.24) (MAB_9229_v114_revA_17Sep2025)
MinION: Protocol
Microbial Amplicon Barcoding Sequencing for 16S and ITS (SQK-MAB114.24) V MAB_9229_v114_revA_17Sep2025
A protocol for amplifying and full-length sequencing of 16S rRNA for bacterial profiling and ITS for fungal profiling from diverse sample types. This kit and method feature:
- Inclusive 16S primers with boosted taxa representation, and/or ITS primers
- Genus-level bacterial identification
- Up to 24 barcodes to multiplex different samples
- A rapid workflow that is faster and more cost-effective than a ligation-based approach
- A fragmentation-free workflow that preserves full-length amplicons
For Research Use Only.
This is an Early Access product.
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
Library preparation
- 3. PCR amplification
- 4. Amplicon barcoding
- 5. Rapid Adapter Attachment
- 6. Priming and loading the MinION and GridION Flow Cell
Sequencing and data analysis
Troubleshooting
Overview
A protocol for amplifying and full-length sequencing of 16S rRNA for bacterial profiling and ITS for fungal profiling from diverse sample types. This kit and method feature:
- Inclusive 16S primers with boosted taxa representation, and/or ITS primers
- Genus-level bacterial identification
- Up to 24 barcodes to multiplex different samples
- A rapid workflow that is faster and more cost-effective than a ligation-based approach
- A fragmentation-free workflow that preserves full-length amplicons
For Research Use Only.
This is an Early Access product.
1. Overview of the protocol
Introduction to the Microbial Amplicon Barcoding Sequencing Kit 24 V14
This protocol describes how to carry out microbial amplicon barcoding sequencing for 16S and ITS using the Microbial Amplicon Barcoding Sequencing Kit 24 V14 (SQK-MAB114.24). Due to the presence of both highly conserved (adequate for universal primers and phylogenetic signal) and highly variant regions (different across species), the 16S rRNA gene can be used for bacterial profiling and ITS for fungal profiling from diverse sample types.
The Microbial Amplicon Barcoding Sequencing Kit 24 V14 includes 16S and ITS primers designed to amplify commonly used regions for microbial identification. This targeted approach focuses sequencing on informative regions, helping you characterise the composition of your samples without sequencing unnecessary parts of the genome. This makes your experiments faster and more cost-effective. The kit also provides 24 unique barcodes, enabling pooling of up to 24 samples in a single run while retaining full-length amplicon information.
After sequencing, you can perform downstream analysis using the EPI2ME 16S workflow (wf-16s) to classify 16S and ITS amplicons from your samples.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your gDNA, and check its length, quantity and purity using the Input DNA/RNA QC protocol. The quality checks performed during the protocol are essential in ensuring experimental success
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- Check your flow cell to ensure it has enough pores for a good sequencing run
Library preparation
The table below is an overview of the steps required in the library preparation, including timings and stopping points.
| Library preparation step | Process | Time | Stop option |
|---|---|---|---|
| 16S or ITS PCR amplification | Amplify the 16S or ITS gene using the primers supplied in the kit | 10 minutes + ~90 minute PCR | 4°C overnight |
| Amplicon barcoding | Attach barcodes to up to 24 amplicon samples | 15 minutes | |
| Barcode inactivation, sample pooling and bead clean-up | Inactivate the barcoding reaction, pool your barcoded samples and perform a sample clean-up | 40 minutes | 4°C short-term storage or for repeated use, such as re-loading your flow cell. -80°C for single-use long-term storage. |
| Adapter ligation | Attach the rapid sequencing adapters to the to the DNA ends. | 5 minutes | We strongly recommend sequencing your library as soon as it is adapted. |
| Priming and loading the flow cell | Prime the flow cell and load the prepared DNA library for sequencing | 5 minutes |
Sequencing and analysis
You will need to:
- Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
- Optional: Start the EPI2ME software and select the wf-16S workflow
Compatibility of this protocol
This protocol should only be used in combination with:
- Microbial Amplicon Barcoding Sequencing Kit 24 V14 (SQK-MAB114.24)
- R10.4.1 flow cells (FLO-MIN114)
- Flow Cell Wash Kit (EXP-WSH004)
- Rapid Adapter Auxiliary V14 (EXP-RAA114)
- Sequencing Auxiliary Vials V14 (EXP-AUX003)
- Flow Cell Priming Kit V14 (EXP-FLP004)
- MinION Mk1B - MinION Mk1B IT requirements document
- MinION Mk1C - MinION Mk1C IT requirements document
- MinION Mk1D - MinION Mk1D IT requirements document
- GridION - GridION IT requirements document
2. Equipment and consumables
Materials
- 10 ng genomic DNA per sample
- Microbial Amplicon Barcoding Sequencing Kit 24 V14 (SQK-MAB114.24)
Consumables
- MinION/GridION Flow Cell
- NEBNext® Thermolabile Proteinase K (E7362)
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 0.2 ml thin-walled PCR tubes
Equipment
- MinION or GridION device
- MinION/GridION Flow Cell Light Shield
- Hula mixer (gentle rotator mixer)
- Microfuge
- Vortex mixer
- Magnetic separation rack, suitable for 1.5 ml Eppendorf tubes
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Multichannel pipette and tips
- Ice bucket with ice
- Timer
- Qubit™ fluorometer (or equivalent for QC check)
For this protocol, you will need 10 ng high molecular weight genomic DNA per sample for amplification.
Third-party reagents
We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.
For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.
Check your flow cell
We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for MinION/GridION/PromethION Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.
| Flow cell | Minimum number of active pores covered by warranty |
|---|---|
| MinION/GridION Flow Cell | 800 |
| PromethION Flow Cell | 5000 |
Microbial Amplicon Barcoding Sequencing Kit 24 V14 contents
| 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.
3. PCR amplification
Materials
- 10 ng genomic DNA per sample
- 16S Primers
- ITS Primers
Consumables
- LongAmp Hot Start Taq 2X Master Mix (NEB, M0533)
- Qubit™ dsDNA HS Assay Kit (ThermoFisher, Q32851)
- Nuclease-free water (e.g. ThermoFisher, AM9937) (chilled)
- 1.5 ml Eppendorf microcentrifuge tubes
- 0.2 ml thin-walled PCR tubes
Equipment
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Microfuge
- Thermal cycler and/or heating block
- 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
Check your flow cell.
We recommend performing a flow cell check before starting your library prep to ensure you have a flow cell with enough pores for a good sequencing run.
See the flow cell check instructions in the MinKNOW protocol for more information.
Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.
| Reagent | 1. Thaw at room temperature | 2. Briefly spin down | 3. Mix well by pipetting |
|---|---|---|---|
| 16S Primers | ✓ | ✓ | ✓ |
| ITS Primers | ✓ | ✓ | ✓ |
| LongAmp Hot Start Taq 2X Master Mix | ✓ | ✓ | ✓ |
Keep your 16S and ITS samples separate.
Please ensure you use the correct primer for your sample type.
Note: For optimal results we recommend sequencing only the same type of amplicons (e.g. 16S amplicons only or ITS amplicons only) on the same flow cell.
Prepare the DNA in nuclease-free water.
- Transfer 10 ng of each genomic DNA sample into a separate 0.2 ml thin-walled PCR tube
- Adjust the volume to 20 μl with nuclease-free water
- Mix thoroughly by flicking avoiding unwanted shearing
- Spin down briefly in a microfuge
In each 0.2 ml thin-walled PCR tube containing a sample to be tested, prepare the following mixture:
For 16S samples:
| Reagent | Volume |
|---|---|
| 10 ng input DNA (from previous step) | 20 μl |
| 16S Primers | 5 μl |
| LongAmp Hot Start Taq 2X Master Mix | 25 μl |
| Total | 50 μl |
Note: If the amount of input material is altered, the number of PCR cycles may need to be adjusted to produce the same yield.
For ITS samples:
| Reagent | Volume |
|---|---|
| 10 ng input DNA (from previous step) | 20 μl |
| ITS Primers | 5 μl |
| LongAmp Hot Start Taq 2X Master Mix | 25 μl |
| Total | 50 μl |
Note: If the amount of input material is altered, the number of PCR cycles may need to be adjusted to produce the same yield.
Ensure the components are thoroughly mixed by pipetting and spin down briefly.
Amplify using the following cycling conditions:
| Cycle step | Temperature | Time | No. of cycles |
|---|---|---|---|
| Initial denaturation | 95 °C | 1 minute | 1 |
| Denaturation Annealing Extension | 95 °C 55 °C 65 °C | 20 seconds 30 seconds 2 minutes | 25 |
| Final extension | 65 °C | 5 minutes | 1 |
| Hold | 4 °C | ∞ |
Remove your amplified samples from your thermal cycler, spin them down briefly, and pipette mix each of them using a fresh pipette tip each time.
Quantify 1 µl of each amplified sample using a Qubit fluorometer (or equivalent) for QC check.
Take forward your amplified sample(s) to the barcoding step of this protocol.
At this stage your amplified sample(s) can be stored short-term at 4°C.
4. Amplicon barcoding
Materials
- 10 ng of each amplicon sample (from previous step)
- OR 20 ng of each amplicon sample (from previous step) if using fewer than 12 Amplicon Barcodes
- Amplicon Barcodes 01-24 (AB01-24)
- S Fragment Buffer (SFB)
- Elution Buffer (EB)
Consumables
- Thermolabile Proteinase K (e.g. NEB, cat # P8111)
- Qubit™ dsDNA HS Assay Kit (ThermoFisher, Q32851)
- Nuclease-free water (e.g. ThermoFisher, AM9937) (chilled)
- 1.5 ml Eppendorf microcentrifuge tubes
- 0.2 ml thin-walled PCR tubes
Equipment
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Microfuge
- Thermal cycler and/or heating block
- 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
Minimum input requirement for lower numbers of Amplicon Barcode use.
For optimal output, we recommend using 20 ng amplicon input per sample if using fewer than 12 barcodes.
Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.
| Reagent | 1. Thaw at room temperature | 2. Briefly spin down | 3. Mix well by pipetting |
|---|---|---|---|
| Amplicon Barcodes 01-24 (AB01-24) | ✓ | ✓ | ✓ |
| EDTA (EDTA) | ✓ | ✓ | ✓ |
| S Fragment Buffer (SFB) | ✓ | ✓ | ✓ |
| AMPure XP Beads (AXP) | ✓ | ✓ | Mix by pipetting or vortexing immediately before use |
| Elution Buffer (EB) | ✓ | ✓ | ✓ |
| Thermolabile Proteinase K | ✓ | ✓ | ✓ |
Prepare the amplicon DNA sample(s) in nuclease-free water.
- Transfer each amplicon DNA sample (from previous steps) into a separate 0.2 ml thin-walled PCR tube:
| For 12 or more samples | For 11 or less samples |
|---|---|
| Transfer 10 ng of each amplicon DNA sample | Transfer 20 ng of each amplicon DNA sample |
- Adjust the volume to 8 μl with nuclease-free water
- Mix thoroughly by flicking, avoiding unwanted shearing
- Spin down briefly in a microfuge
Select a unique Amplicon Barcode for each sample to be run together on the same flow cell.
Up to 24 samples can be barcoded and combined in one experiment.
Note: Only use one barcode per sample. For optimal results, we recommend running only the same type of amplicons (e.g. 16S amplicons only or ITS amplicons only) on the same flow cell.
In each 0.2 ml thin-walled PCR tube containing your amplicon sample to be tested, prepare the following mixture:
| Reagent | Volume |
|---|---|
| 16S or ITS Amplicon DNA (from previous step) | 8 μl |
| EDTA | 1 μl |
| Amplicon Barcode (AB01-24) | 1 μl |
| Total | 10 μl |
Ensure the reactions are thoroughly mixed by pipetting and spin down.
Incubate the reactions in a thermal cycler at 65°C for 10 minutes, then at 80°C for 2 minutes.
Remove your samples from your thermal cycler and spin them down briefly.
Add 1 ul Thermolabile Proteinase K to each reaction and mix thoroughly by pipetting.
Incubate the reactions in a thermal cycler at 37°C for 15 minutes, then at 55°C for 10 minutes.
Remove your samples from your thermal cycler and spin them down briefly.
Pool all barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
| Volume per sample | For 6 samples | For 12 samples | For 24 samples | |
|---|---|---|---|---|
| Total volume | ~11 μl | 66 μl | 132 μl | 264 μl |
Resuspend the AMPure XP Beads (AXP) by vortexing.
To the pool of barcoded samples, add a 0.7X volume ratio of resuspended AMPure XP Beads (AXP) and mix by pipetting:
| . | Volume per sample | For 6 samples | For 12 samples | For 24 samples |
|---|---|---|---|---|
| Volume of AMPure XP Beads (AXP) | 7.7 μl | 46 μl | 93 μl | 185 μl |
Note: Table contains example volumes for reference. Please adjust the volume of AMPure XP Beads (AXP) added for the volume of your barcoded sample pool to ensure a 0.7X volume ratio.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
Briefly spin down the sample and pellet on a magnetic rack until supernatant is clear and colourless. Keep the tube on the magnetic rack, and pipette off the supernatant.
Wash the beads by adding 250 µl S Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet by pipetting in 15 µl Elution Buffer (EB). Spin down and incubate for 5 minutes at room temperature.
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Remove and retain 15 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
- Remove and retain the eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube
- Dispose of the pelleted beads
Quantify 1 µl of eluted sample using a Qubit fluorometer.
Please note, if your library concentration is very low the DNA library might have been lost during the clean-up steps of the prep. Ensure you have sufficient DNA library to load on your flow cell.
You should expect ≥50% recovery from your total sample input into the amplicon barcoding step of the protocol.
Transfer 11 µl of your eluted sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
Take forward your eluted barcoded DNA library to the rapid adapter attachment step of this protocol.
At this stage your eluted barcoded DNA library can be stored at 4°C for short-term storage. For longer term storage you can store the DNA library at -80°C.
5. Rapid Adapter Attachment
Materials
- DNA library eluate
- Rapid Adapter (RA)
- Adapter Buffer (ADB)
Consumables
- 1.5 ml Eppendorf microcentrifuge tubes
Equipment
- Vortex mixer
- Microfuge
- 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
Thaw the following reagents, then spin down briefly using a microfuge and mix as indicated in the table below. Then place the reagents on ice.
| Reagent | 1. Thaw at room temperature | 2. Briefly spin down | 3. Mix well by pipetting |
|---|---|---|---|
| Rapid Adapter (RA) | ✓ | ✓ | ✓ |
| Adapter Buffer (ADB) | ✓ | ✓ | ✓ |
In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows and pipette mix:
| Reagent | Volume |
|---|---|
| Rapid Adapter (RA) | 1.5 μl |
| Adapter Buffer (ADB) | 3.5 μl |
| Total | 5 μl |
Add 1 µl of the diluted Rapid Adapter (RA) to the barcoded DNA.
Mix gently by flicking the tube, and spin down.
Incubate the reaction for 5 minutes at room temperature.
The prepared library is used for loading into the flow cell. Store the library on ice until ready to load.
6. Priming and loading the MinION and GridION Flow Cell
Materials
- Flow Cell Flush (FCF)
- Flush Tether UL (FTU)
- Library Beads (LIB)
- Sequencing Buffer (SB)
Consumables
- MinION/GridION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
Equipment
- MinION or GridION device
- MinION/GridION Flow Cell Light Shield
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Vortex mixer
Please note, this kit is only compatible with R10.4.1 flow cells (FLO-MIN114).
Priming and loading a flow cell
We recommend all new users watch the 'Priming and loading your flow cell' video before your first run.
Thaw the Sequencing Buffer (SB), Library Beads (LIB), Flush Tether UL (FTU) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.
To prepare the flow cell priming mix, combine Flow Cell Flush (FCF) and Flush Tether UL (FTU), as directed below. Mix by pipetting at room temperature.
In a suitable tube for the number of flow cells, combine the following reagents:
| Reagent | Volume per flow cell |
|---|---|
| Flow Cell Flush (FCF) | 1,197 µl |
| Flush Tether UL (FTU) | 3 µl |
| Total volume | 1,200 µl |
Open the MinION or GridION device lid and slide the flow cell under the clip. Press down firmly on the priming port cover to ensure correct thermal and electrical contact.
Complete a flow cell check to assess the number of pores available before loading the library.
This step can be omitted if the flow cell has been checked previously.
See the flow cell check instructions in the MinKNOW protocol for more information.
Slide the flow cell priming port cover clockwise to open the priming port.
Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
- Set a P1000 pipette to 200 µl
- Insert the tip into the priming port
- Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip
Note: Visually check that there is continuous buffer from the priming port across the sensor array.
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.
Thoroughly mix the contents of the Library Beads (LIB) by pipetting.
The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:
| Reagent | Volume per flow cell |
|---|---|
| Sequencing Buffer (SB) | 37.5 µl |
| Library Beads (LIB) mixed immediately before use, or Library Solution (LIS), if using | 25.5 µl |
| DNA library | 12 µl |
| Total | 75 µl |
Complete the flow cell priming:
- Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
- Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
Mix the prepared library gently by pipetting up and down just prior to loading.
Add 75 μl of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.
We recommend leaving the light shield on the flow cell when library is loaded, including during any washing and reloading steps. The shield can be removed when the library has been removed from the flow cell.
Place the light shield onto the flow cell, as follows:
Carefully place the leading edge of the light shield against the clip. Note: Do not force the light shield underneath the clip.
Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.
The MinION Flow Cell Light Shield is not secured to the flow cell and careful handling is required after installation.
Close the device lid and set up a sequencing run on MinKNOW.
When a flow cell is inserted into the MinION Mk1D, the device lid will sit on top of the flow cell, leaving a small gap around the sides. This is normal and has no impact on the performance of the device.
Please refer to this FAQ regarding the device lid.
7. Data acquisition and basecalling
How to start sequencing
Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.
MinKNOW can be used and set up to sequence in multiple ways:
- On a computer either directly or remotely connected to a sequencing device.
- Directly on a GridION or PromethION 24/48 sequencing device.
For more information on using MinKNOW on a sequencing device, please see the device user manuals:
- MinION Mk1B user manual
- MinION Mk1D user manual
- GridION user manual
- PromethION user manual
- PromethION 2 Solo user manual
To start a sequencing run on MinKNOW:
1. Navigate to the start page and click Start sequencing.
2. Fill in your experiment details, such as name and flow cell position and sample ID.
3. Select the sequencing kit used in the library preparation on the Kit page.
4. Configure the sequencing and output parameters for your sequencing run or keep to the default settings on the Run configuration tab.
Note: If basecalling was turned off when a sequencing run was set up, basecalling can be performed post-run on MinKNOW. For more information, please see the MinKNOW protocol.
5. Click Start to initiate the sequencing run.
Data analysis after sequencing
After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.
After sequencing and basecalling, the data can be analysed. For further information about options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.
In the Downstream analysis section, we outline further options for analysing your data.
8. Flow cell reuse and returns
Materials
- Flow Cell Wash Kit (EXP-WSH004)
After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Flow Cell Wash Kit protocol and store the washed flow cell at +2°C to +8°C.
The Flow Cell Wash Kit protocol is available on the Nanopore Community.
Alternatively, follow the returns procedure to send the flow cell back to Oxford Nanopore.
Instructions for returning flow cells can be found here.
If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting Guide that can be found in the online version of this protocol.
9. Downstream analysis
Post-basecalling analysis
We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a desktop application. EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence analysis.
Further information about the available EPI2ME workflows are available here, along with the Quick Start Guide to start your first bioinformatic workflow.
The 16S workflow (wf-16s) is a Nextflow workflow leveraging the power of wf-metagenomics for identification of the origin of reads from targeted amplicon sequencing. The workflow has two modes of operation, it can use either kraken2 or minimap2 to determine the origin of reads.
More information on the EPI2ME 16S workflow (wf-16s) can be found here.
For installation instructions please click here.
Additional options for further analysing your basecalled data include:
1. Research analysis tools
Oxford Nanopore Technologies' Research division has created a number of analysis tools, that are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.
2. Community-developed analysis tools
If a data analysis method for your research question is not provided in any of the resources above, please refer to the Bioinformatics section of the Resource centre. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.
10. Issues during DNAextraction and library preparation
Below is a list of the most commonly encountered issues, with some suggested causes and solutions.
We also have an FAQ section available on the Nanopore Community Support section.
If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.
Low sample quality
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Low DNA purity (Nanodrop reading for DNA OD 260/280 is <1.8 and OD 260/230 is <2.0–2.2) | The DNA extraction method does not provide the required purity | The effects of contaminants are shown in the Contaminants document. Please try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional SPRI clean-up step. |
Low DNA recovery after AMPure bead clean-up
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Low recovery | DNA loss due to a lower than intended AMPure beads-to-sample ratio | 1. AMPure beads settle quickly, so ensure they are well resuspended before adding them to the sample. 2. When the AMPure beads-to-sample ratio is lower than 0.4:1, DNA fragments of any size will be lost during the clean-up. |
| Low recovery | DNA fragments are shorter than expected | The lower the AMPure beads-to-sample ratio, the more stringent the selection against short fragments. Please always determine the input DNA length on an agarose gel (or other gel electrophoresis methods) and then calculate the appropriate amount of AMPure beads to use.  |
| Low recovery after end-prep | The wash step used ethanol <80% | DNA will be eluted from the beads when using ethanol <80%. Make sure to use the correct percentage. |
11. Issues during the sequencing run using a Rapid-based sequencing kit
Below is a list of the most commonly encountered issues, with some suggested causes and solutions.
We also have an FAQ section available on the Nanopore Community Support section.
If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.
Fewer pores at the start of sequencing than after Flow Cell Check
| Observation | Possible cause | Comments and actions |
|---|---|---|
| MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check | An air bubble was introduced into the nanopore array | After the Flow Cell Check it is essential to remove any air bubbles near the priming port before priming the flow cell. If not removed, the air bubble can travel to the nanopore array and irreversibly damage the nanopores that have been exposed to air. The best practice to prevent this from happening is demonstrated in this video. |
| MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check | The flow cell is not correctly inserted into the device | Stop the sequencing run, remove the flow cell from the sequencing device and insert it again, checking that the flow cell is firmly seated in the device and that it has reached the target temperature. If applicable, try a different position on the device (GridION/PromethION). |
| MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check | Contaminations in the library damaged or blocked the pores | The pore count during the Flow Cell Check is performed using the QC DNA molecules present in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate the number of active pores. Because of this, variability of about 10% in the number of pores is expected. A significantly lower pore count reported at the start of sequencing can be due to contaminants in the library that have damaged the membranes or blocked the pores. Alternative DNA/RNA extraction or purification methods may be needed to improve the purity of the input material. The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover. |
MinKNOW script failed
| Observation | Possible cause | Comments and actions |
|---|---|---|
| MinKNOW shows "Script failed" | Restart the computer and then restart MinKNOW. If the issue persists, please collect the MinKNOW log files and contact Technical Support. If you do not have another sequencing device available, we recommend storing the flow cell and the loaded library at 4°C and contact Technical Support for further storage guidance. |
Pore occupancy below 40%
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Pore occupancy <40% | Not enough library was loaded on the flow cell | 10–50 fmol of good quality library can be loaded on to a MinION Mk1B/GridION flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol" |
| Pore occupancy close to 0 | The Rapid PCR Barcoding Kit V14 was used, and sequencing adapters did not attach to the DNA | Make sure to closely follow the protocol and use the correct volumes and incubation temperatures. A Lambda control library can be prepared to test the integrity of reagents. |
| Pore occupancy close to 0 | No tether on the flow cell | Tethers are added during flow cell priming (FCT tube). Make sure FCT was added to FCF before priming. |
Shorter than expected read length
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Shorter than expected read length | Unwanted fragmentation of DNA sample | Read length reflects input DNA fragment length. Input DNA can be fragmented during extraction and library prep. 1. Please review the Extraction Methods in the Nanopore Community for best practice for extraction. 2. Visualise the input DNA fragment length distribution on an agarose gel before proceeding to the library prep.  In the image above, Sample 1 is of high molecular weight, whereas Sample 2 has been fragmented.3. During library prep, avoid pipetting and vortexing when mixing reagents. Flicking or inverting the tube is sufficient. |
Large proportion of unavailable pores
| Observation | Possible cause | Comments and actions |
|---|---|---|
Large proportion of unavailable pores (shown as blue in the channels panel and pore activity plot)  The pore activity plot above shows an increasing proportion of "unavailable" pores over time. | Contaminants are present in the sample | Some contaminants can be cleared from the pores by the unblocking function built into MinKNOW. If this is successful, the pore status will change to "sequencing pore". If the portion of unavailable pores stays large or increases: 1. A nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) can be performed, or 2. Run several cycles of PCR to try and dilute any contaminants that may be causing problems. |
Large proportion of inactive pores
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Large proportion of inactive/unavailable pores (shown as light blue in the channels panel and pore activity plot. Pores or membranes are irreversibly damaged) | Air bubbles have been introduced into the flow cell | Air bubbles introduced through flow cell priming and library loading can irreversibly damage the pores. Watch the Priming and loading your flow cell video for best practice |
| Large proportion of inactive/unavailable pores | Certain compounds co-purified with DNA | Known compounds, include polysaccharides, typically associate with plant genomic DNA. 1. Please refer to the Plant leaf DNA extraction method. 2. Clean-up using the QIAGEN PowerClean Pro kit. 3. Perform a whole genome amplification with the original gDNA sample using the QIAGEN REPLI-g kit. |
| Large proportion of inactive/unavailable pores | Contaminants are present in the sample | The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover. |
Temperature fluctuation
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Temperature fluctuation | The flow cell has lost contact with the device | Check that there is a heat pad covering the metal plate on the back of the flow cell. Re-insert the flow cell and press it down to make sure the connector pins are firmly in contact with the device. If the problem persists, please contact Technical Services. |
Failed to reach target temperature
| Observation | Possible cause | Comments and actions |
|---|---|---|
| MinKNOW shows "Failed to reach target temperature" | The instrument was placed in a location that is colder than normal room temperature, or a location with poor ventilation (which leads to the flow cells overheating) | MinKNOW has a default timeframe for the flow cell to reach the target temperature. Once the timeframe is exceeded, an error message will appear and the sequencing experiment will continue. However, sequencing at an incorrect temperature may lead to a decrease in throughput and lower q-scores. Please adjust the location of the sequencing device to ensure that it is placed at room temperature with good ventilation, then re-start the process in MinKNOW. Please refer to this link for more information on MinION temperature control. |
12. Primer design, PCR and sequencing considerations
16S and ITS rRNA sequences
A full list of the primer sequences for both the 16S primers (16S) and ITS primers (ITS) can be found in this downloadable document:
Our primer schemes are aligned with industry standards 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.
ITS primer design
The ITS primers supplied in this kit are aligned with industry standards.
Please note, the ITS1 primer can form dimers, leading to a small number of PCR artefacts that will be carried through the library preparation. This may produce a small population of longer or lower-quality reads (concatemer reads or 2D reads with supplementary alignments). These reads are automatically filtered in our EPI2ME workflow using minimap2, but if you use other analysis tools, please account for them.
16S primer design
The 16S primers supplied in this kit have not presented any behaviour conducive with dimerisation and the formation of hairpins.
Last updated: 9/17/2025
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