Oct 07, 2025
Version 6
Nanopore amplicon sequencing V.6
- Yoshiyuki Matsuo1
- 1Kansai Medical University
External link: https://doi.org/10.1186/s12920-022-01218-8
Protocol Citation: Yoshiyuki Matsuo 2025. Nanopore amplicon sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5zrodv1b/v6Version created by Yoshiyuki Matsuo
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2025
Last Modified: October 07, 2025
Protocol Integer ID: 228909
Keywords: amplicon sequencing, long read, nanopore, MinION, performing nanopore amplicon, nanopore amplicon, human microbiome profiling, using environmental dna, including human microbiome profiling, environmental dna, biodiversity assessment, kit 14 chemistry, latest kit 14 chemistry, amplicon, sequence
Abstract
This protocol describes the step-by-step procedures for performing nanopore amplicon sequencing using the latest Kit 14 chemistry. The method is versatile and can be applied to a wide range of sequence-based analyses, including human microbiome profiling and biodiversity assessment using environmental DNA (eDNA metabarcoding).
Materials
Reagents and kits
- DNA sample
- Tailed PCR primers (target-specific primers with anchor sequences at the 5' ends)
- PrimeSTAR Max DNA Polymerase Ver.2 (TaKaRa, R047A)
- PCR Barcoding Expansion 1–12 (Oxford Nanopore Technologies, EXP-PBC001) or PCR Barcoding Expansion 1–96 (Oxford Nanopore Technologies, EXP-PBC096)
- ProNex Size-Selective Purification System (Promega, NG2001)
- 80% ethanol
- QuantiFluor ONE dsDNA System (Promega, E4871)
- NEBNext Ultra II End Repair/dA-tailing Module (New England Biolabs, E7546S)
- Ligation Sequencing Kit V14 (Oxford Nanopore Technologies, SQK-LSK114)
- Salt-T4 DNA Ligase (New England Biolabs, M0467S)
- UltraPure BSA (50 mg/mL) (Thermo Fisher Scientific, AM2616)
- Flow Cell Wash Kit (Oxford Nanopore Technologies, EXP-WSH004)
Equipments
- Thermal cycler
- Gel electrophoresis apparatus
- Magnetic separation rack
- Microcentrifuge
- Vortex mixer
- Quantus Fluorometer (Promega, E6150)
- MinION Mk1D (Oxford Nanopore Technologies, MIN-101D)
- MinION Flow Cell R10.4.1 (Oxford Nanopore Technologies, FLO-MIN114)
- Apple Silicon Mac
Protocol materials
NEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
PCR Barcoding Expansion 1–12Oxford Nanopore TechnologiesCatalog #EXP-PBC001
PCR Barcoding Expansion 1–96Oxford Nanopore TechnologiesCatalog #EXP-PBC096
PrimeSTAR Max DNA Polymerase Ver.2Takara Bio Inc.Catalog #R047A
QuantiFluor ONE dsDNA SystemPromegaCatalog #E4871
Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
Salt-T4 DNA LigaseNew England BiolabsCatalog #M0467S
Flow Cell Wash KitOxford Nanopore TechnologiesCatalog #EXP-WSH004
ProNex Size-Selective Purification SystemPromegaCatalog #NG2001
UltraPure BSA (50 mg/mL)Thermo Fisher ScientificCatalog #AM2616
Troubleshooting
Workflow
In the first-round PCR reaction, the region of interest is amplified using specific primers flanked by anchor sequences. The tailed PCR products are then subjected to a second round of PCR to incorporate barcode sequences for multiplexing. The barcoded amplicons are pooled and end-prepped, allowing for the ligation of sequencing adapters.
First-round PCR with tailed primers
Prepare the PCR master mix.
| A | B | C | |
| Component | Volume | Final conc. | |
| Template DNA | x µL | ||
| 5 µM FW/RV primer mix | 1.5–2.5 µL | 0.3–0.5 µM each | |
| PrimeSTAR Max Ver.2 Premix (2X) | 12.5 µL | 1X | |
| Water | up to 25 µL |
PrimeSTAR Max DNA Polymerase Ver.2Takara Bio Inc.Catalog #R047A
Tailed primers (user-supplied)
| A | B | |
| Primer | Sequence | |
| Forward (FW) | 5'-TTTCTGTTGGTGCTGATATTGC - target-specific sequence -3' | |
| Reverse (RV) | 5'-ACTTGCCTGTCGCTCTATCTTC - target-specific sequence -3' |
The 5' anchor sequences serve as priming sites for barcoded primers used in the 2nd PCR.
Note
The following tailed primers are used for amplifying the V1–V9 region of the bacterial 16S rRNA gene. 16S rRNA gene-specific sequences are in bold letters.
- 27F*:
5'-TTTCTGTTGGTGCTGATATTGC AGRGTTYGATYMTGGCTCAG-3'
- 1492R:
5'-ACTTGCCTGTCGCTCTATCTTC CRGHTACCTTGTTACGACTT-3'
*We observed that the 27F universal primer described in Protocol Version 5 exhibits reduced amplification efficiency and specificity, likely due to its increased degeneracy. To avoid these issues, the primer used in the previous version of the protocol, as described above, is recommended.
Perform PCR.
| A | B | C | D | |
| Step | Temperature | Time | Cycles | |
| Denaturation | 98°C | 10 sec | 25–35 | |
| Annealing | 57°C | 15 sec | ||
| Extension | 68°C | 5–10 sec/kb* | ||
| Hold | 4°C | ∞ | 1 |
*e.g. Use an extension time of 10 seconds per cycle to amplify the near-full length (V1–V9 regions) of bacterial 16S rRNA genes (approximately 1.5 kbp), using the high-speed PrimeSTAR DNA polymerase.
Analyze 1–2 µL of the PCR products by gel electrophoresis to verify successful amplification.
PCR clean-up (Optional)
Allow the ProNex beads to equilibrate to room temperature.
Note
The first-round PCR products may need to be purified before proceeding to the second-round of PCR. This additional step removes reaction contaminants, which would be beneficial for the downstream analysis.
ProNex Size-Selective Purification SystemPromegaCatalog #NG2001
Add ProNex beads to the first-round PCR products and mix by pipetting.
| A | B | |
| Component | Volume | |
| First-round PCR products | 20 µL | |
| ProNex Chemistry | 20 µL |
Note
The ratio of ProNex beads to sample varies depending on the desired DNA size cutoff range. The condition shown above will enrich DNA fragments larger than 1 kbp using a 1:1 ratio of ProNex beads to sample.
As an alternative to ProNex beads, AMPure XP can also be used. The ratio of AMPure beads to the sample should be adjusted accordingly.
Incubate at Room temperature for 00:05:00 .
Place the tube on a magnetic rack for 00:02:00 .
Equipment
NGS MagnaStand v.3 8Ch
NAME
Magnetic rack (0.2 mL tube)
TYPE
FastGene
BRAND
FG-SSMAG3
SKU
Pipette off the supernatant.
Wash the beads once with 80% ethanol using the following procedure:
Keeping on the magnetic rack, add 200 µL of 80% ethanol without disturbing the bead pellet.
Discard the supernatant.
Spin down the tube and place it back on the magnetic rack.
Pipette off any residual ethanol.
Remove the tube from the magnetic rack and allow the sample to air-dry for ~2 min.
Resuspend the beads in 20 µL of water.
Incubate at Room temperature for 00:05:00 .
Place the tube on the magnetic rack for 00:02:00 .
Transfer the eluate to a new tube.
Second-round PCR with barcoded primers
Prepare the PCR master mix.
| A | B | |
| Component | Volume | |
| First-round PCR products | 1.0 µL | |
| PCR Barcode (BC)* | 0.5 µL | |
| PrimeSTAR Max Ver.2 Premix (2X) | 12.5 µL | |
| Water | 11 µL | |
| Total | 25 µL |
*One of barcoded primers (10 µM BC) supplied in the PCR Barcoding Expansion (EXP-PBC001 or EXP-PBC096).
PCR Barcoding Expansion 1–12Oxford Nanopore TechnologiesCatalog #EXP-PBC001
PCR Barcoding Expansion 1–96Oxford Nanopore TechnologiesCatalog #EXP-PBC096
Perform PCR to incorporate barcode sequences.
| A | B | C | D | |
| Step | Temperature | Time | Cycles | |
| Denaturation | 98°C | 10 sec | 5–10 | |
| Annealing | 57°C | 15 sec | ||
| Extension | 68°C | 5–10 sec/kb* | ||
| Hold | 4°C | ∞ | 1 |
*e.g. Use an extension time of 10 seconds per cycle to amplify the near-full length (V1–V9 regions) of bacterial 16S rRNA genes (approximately 1.5 kbp), using the high-speed PrimeSTAR DNA polymerase.
Analyze 1–2 µL of the PCR products by gel electrophoresis.
PCR clean-up
14m
Allow the ProNex beads to equilibrate to room temperature.
Pool the barcoded amplicons into a single tube.
Note
As an example, when working with 10 barcodes, pool 10 µL from each sample to obtain a final volume of 100 µL; alternatively, the samples can be pooled in desired ratios, and the total volume can be adjusted flexibly depending on the experimental requirements.
Each sample may also be cleaned up separately prior to pooling. After quantifying the DNA, the samples can be pooled in the desired ratios to balance input amounts and achieve uniform sequencing data, and subsequently subjected to the End-prep (step 41).
Add ProNex beads to the pooled sample and mix by pipetting.
| A | B | |
| Component | Volume | |
| Pooled sample | 100 µL | |
| ProNex Chemistry | 100 µL |
This is an example protocol based on an input sample volume of 100 µL.
Note
The ratio of ProNex beads to sample varies depending on the desired DNA size cutoff range. The condition shown above will enrich DNA fragments larger than 1 kbp using a 1:1 ratio of ProNex beads to sample.
As an alternative to ProNex beads, AMPure XP can also be used. The ratio of AMPure beads to the sample should be adjusted accordingly.
Incubate at Room temperature for 00:05:00 .
5m
Place the tube on a magnetic rack for 00:02:00 .
2m
Pipette off the supernatant.
Wash the beads with 80% ethanol as follows:
Keeping on the magnetic rack, add 200 µL of 80% ethanol without disturbing the bead pellet.
Discard the supernatant.
Repeat steps 27.1 and 27.2 for a total of two washes.
Spin down the tube and place it back on the magnetic rack.
Pipette off any residual ethanol.
Remove the tube from the magnetic rack and allow the sample to air-dry for ~2 min.
Resuspend the beads in 26 µL of water.
Incubate at Room temperature for 00:05:00 .
5m
Place the tube on the magnetic rack for 00:02:00 .
2m
Transfer the eluate to a new tube.
DNA quantification
Warm QuantiFluor ONE dsDNA dye to Room temperature .
QuantiFluor ONE dsDNA SystemPromegaCatalog #E4871
Add 1 µL of eluted sample to 200 µL of QuantiFluor ONE dsDNA dye in 0.5 mL tube.
Mix thoroughly by vortexing.
Incubate at Room temperature for 00:05:00 , protected from light.
5m
Measure fluorescence using the Quantus Fluorometer to quantify DNA concentration.
Equipment
Quantus Fluorometer
NAME
Promega
BRAND
E6150
SKU
End-prep
Prepare 200 fmol of pooled DNA (from step 35) in 25 µL of water.
Note
For full-length 16S rRNA gene sequencing, 200 fmol of 1.5 kbp amplicons correspond to approximately 200 ng in weight.
Mix the following components.
| A | B | |
| Component | Volume | |
| Pooled amplicon DNA | 25 µL | |
| NEBNext Ultra II End Prep Reaction Buffer | 3.5 µL | |
| NEBNext Ultra II End Prep Enzyme Mix | 1.5 µL | |
| Total | 30 µL |
NEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
Incubate the reaction using a thermal cycler.
| A | B | |
| Temperature | Time | |
| 20°C | 5 min | |
| 65°C | 5 min | |
| 4°C | ∞ |
Note
Amplicons are converted into 5ʹ-phosphorylated and 3ʹ-dA-tailed DNA, enabling ligation of Nanopore sequencing adapters.
Clean-up
14m
Allow the ProNex beads to equilibrate to room temperature.
Add ProNex beads to the end-prep reaction and mix by pipetting.
| A | B | |
| Component | Volume | |
| End-prepped DNA | 30 µL | |
| ProNex Chemistry | 30 µL |
Note
The ratio of beads to sample volume should be adjusted according to the desired DNA fragment size. As an alternative to ProNex beads, AMPure XP can also be used.
Incubate at Room temperature for 00:05:00 .
5m
Place the tube on a magnetic rack for 00:02:00 .
2m
Pipette off the supernatant.
Wash the beads with 80% ethanol as follows:
Keeping on the magnetic rack, add 200 µL of 80% ethanol without disturbing the bead pellet.
Discard the supernatant.
Repeat steps 49.1 and 49.2 for a total of two washes.
Spin down the tube and place it back on the magnetic rack.
Pipette off any residual ethanol.
Remove the tube from the magnetic rack and allow the sample to air-dry for ~2 min.
Resuspend the beads in 30 µL of water.
Incubate at Room temperature for 00:05:00 .
5m
Place the tube on the magnetic rack for 00:02:00 .
2m
Transfer the eluate to a new tube.
Adapter ligation
10m
Mix the following components.
| A | B | |
| Component | Volume | |
| End-prepped DNA | 30 µL | |
| Ligation Buffer (LNB)* | 12.5 µL | |
| Ligation Adapter (LA) | 2.5 µL | |
| Salt-T4 DNA Ligase | 5 µL | |
| Total | 50 µL |
The Ligation Buffer (LNB) and Ligation Adapter (LA) are included in the Ligation Sequencing Kit V14.
*LNB is viscous and should be thoroughly mixed by pipetting.
Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
Salt-T4 DNA LigaseNew England BiolabsCatalog #M0467S
Incubate at Room temperature for 00:10:00 .
10m
Clean-up
12m
Allow the ProNex beads to equilibrate to room temperature.
Add ProNex beads to the adapter-ligated sample and mix by pipetting.
| A | B | |
| Component | Volume | |
| Adapter-ligated DNA | 50 µL | |
| ProNex Chemistry | 50 µL |
Note
The ratio of beads to sample volume should be adjusted according to the desired DNA fragment size. As an alternative to ProNex beads, AMPure XP can also be used.
Incubate at Room temperature for 00:05:00 .
5m
Place the tube on a magnetic rack for 00:02:00 .
Pipette off the supernatant.
Wash the beads with either Short Fragment Buffer (SFB) or Long Fragment Buffer (LFB) as follows:
Note
The Short Fragment Buffer (SFB) and Long Fragment Buffer (LFB) are included in the Ligation Sequencing Kit V14.
For full-length 16S rRNA gene amplicons (approximately 1.5 kbp), use SFB to retain DNA fragments of all sizes. LFB should be used to enrich for DNA fragments larger than 3 kbp.
Remove the tube from the magnetic rack and resuspend the beads in 100 µL of either SFB or LFB.
Place the tube on the magnetic rack for 00:02:00 .
2m
Discard the supernatant.
Repeat steps 65.1–65.3 for a total of two washes.
Spin down the tube and place it back on the magnetic rack.
Pipette off any residual wash buffer.
Remove the tube from the magnetic rack and allow the sample to air-dry for ~2 min.
Resuspend the beads in 15 µL of Elution Buffer (EB).
Note
The Elution Buffer (EB) is included in the Ligation Sequencing Kit V14.
Incubate at Room temperature for 00:05:00 .
5m
Place the tube on the magnetic rack for 00:02:00 .
Transfer the eluate to a new tube.
DNA quantification
Warm QuantiFluor ONE dsDNA dye to Room temperature .
Add 1 µL of eluted sample to 200 µL of QuantiFluor ONE dsDNA dye in 0.5 mL tube.
Mix thoroughly by vortexing.
Incubate at Room temperature for 00:05:00 , protected from light.
Measure fluorescence using the Quantus Fluorometer to quantify DNA concentration.
Flow cell check
Connect the MinION sequencer to the host computer and start the MinKNOW software.
Equipment
MinION Mk1D
NAME
Sequencer
TYPE
Oxford Nanopore Technologies
BRAND
MIN-101D
SKU
Open the MinION lid and insert a flow cell (R10.4.1) under the clip.
Equipment
MinION Flow Cell (R10.4.1)
NAME
Oxford Nanopore Technologies
BRAND
FLO-MIN114
SKU
Perform flow cell check.
Check the number of active pores available for the experiment.
Remove the flow cell from the MinION device and return it to the tray.
Flow cell priming
5m
Prepare the flow cell priming mix.
| A | B | |
| Component | Volume | |
| Flow Cell Tether (FCT) | 30 µL | |
| 50 mg/mL Bovine Serum Albumin (BSA) | 5 µL | |
| Flow Cell Flush (FCF) | 1,170 µL | |
| Total | 1,205 µL |
The Flow Cell Tether (FCT) and Flow Cell Flush (FCF) are included in the Ligation Sequencing Kit V14.
UltraPure BSA (50 mg/mL)Thermo Fisher ScientificCatalog #AM2616
Open the priming port cover of the flow cell.
If any air bubbles are present under the cover, remove them using the following procedure:
Set the volume of P1000 micropipette to 200 µL.
Insert the pipette tip into the priming port.
Turn the wheel of the pipette slowly to increase the volume and draw back 20–30 µL of the buffer.
Note
Care must be taken not to remove too much, ensuring that the sensor array of the flow cell remains covered by the buffer.
Load the priming mix into the flow cell through the priming port as follows:
Note
Avoid introducing air.
Using a P1000 micropipette, aspirate 800 µL of the priming mix.
Insert the pipette tip into the priming port.
Slowly turn the pipette wheel down to load the priming mix into the flow cell.
Wait for 00:05:00 .
5m
Lift the SpotON sample port cover upwards to uncover the sample port on the flow cell.
Load the remaining 200 µL of the priming mix into the priming port (caution: not the SpotON sample port) by slowly turning the pipette wheel down to dispense the mix and complete the flow cell priming.
Sample loading
For 1–10 kbp amplicons, prepare 35–50 fmol of DNA library (from step 73) in 12 µL of Elution Buffer (EB).
Note
For full-length 16S rRNA gene sequencing, 35–50 fmol of 1.5 kbp amplicons correspond to approximately 35–50 ng in weight.
Prepare the sequencing library for loading.
| A | B | |
| Component | Volume | |
| DNA library | 12 µL | |
| Sequencing Buffer (SB) | 37.5 µL | |
| Library Beads (LIB)* | 25.5 µL | |
| Total | 75 µL |
The Sequencing Buffer (SB) and Library Beads (LIB) are included in the Ligation Sequencing Kit V14.
*Since LIB settle quickly, the bead suspension should be mixed immediately before adding it to the loading mixture.
Gently mix the sequencing library by pipetting just prior to loading.
Load 75 µL of the sequencing library into the flow cell via the SpotON sample port in a dropwise fashion.
Note
Using a P100 or P200 micropipette, let each drop flow into the sample port before adding the next one.
Gently lower the SpotON sample port cover back, ensuring the bung enters the sample port.
Close the priming port
Insert the flow cell into the MinION device.
Place the light shield onto the flow cell.
Close the MinION lid.
Nanopore sequencing
Set up a sequencing run on the MinKNOW software.
| A | B | |
| Parameter | Setting | |
| Flow cell type | FLO-MIN114 (R10.4.1) | |
| Kit | Ligation Sequencing Kit SQK-LSK114 | |
| Expansion pack | PCR Barcoding Expansion 1–12 EXP-PBC001 | |
| Basecalling | On | |
| Basecalling model | Fast basecalling | |
| Barcoding | On | |
| Trim barcodes | On | |
| Barcode both ends | On | |
| Q score filtering | 8 (default value) | |
| Min/Max read length* | Optional |
Above are typical examples of run parameters for real-time basecalling on the MinION Mk1D, connected to an Apple Silicon Mac.
*For full-length 16S rRNA gene sequencing, we set the minimum read length to 1,300 bases and the maximum read length to 1,800 bases for a passing read.
Start a sequencing run.
Flow cell washing and storage for reuse
1h 5m
Stop the sequencing run.
Remove the flow cell from the MinION device and place it in the tray.
Using a P1000 micropipette, aspirate the fluid from the waste channel through the waste port.
Note
Ensure that both the priming port and SpotON sample port are closed.
Prepare the flow cell wash mix and gently mix by pipetting.
| A | B | |
| Component | Volume | |
| Wash Mix (WMX)* | 2 µL | |
| Wash Diluent (DIL) | 398 µL | |
| Total | 400 µL |
*WMX contains a nuclease, DNase I, which digests the DNA library.
Flow Cell Wash KitOxford Nanopore TechnologiesCatalog #EXP-WSH004
Open the priming port cover of the flow cell.
[Optional] If necessary, remove air bubbles under the cover by following the procedure in step 86.
Load the wash mix into the flow cell via the priming port as follows:
Note
Avoid introducing air.
Using a P1000 micropipette, aspirate 200 µL of the wash mix.
Insert the pipette tip into the priming port.
Slowly turn the pipette wheel down to load the wash mix into the flow cell.
Wait for 00:05:00 .
5m
Load the remaining 200 µL of the wash mix into the priming port by following the procedure in steps 108.1–108.3.
Close the priming port.
Wait for 01:00:00 to digest the residual DNA on the flow cell.
1h
Open the priming port cover of the flow cell.
[Optional] If necessary, remove air bubbles under the cover by following the procedure in step 86.
Using a P1000 micropipette, load 500 µL of Storage Buffer (S) into the flow cell via the priming port by slowly turning the pipette wheel down.
Note
Avoid introducing air.
Close the priming port.
Using a P1000 micropipette, aspirate the fluid from the waste channel through the waste port.
Note
Ensure that both the priming port and SpotON sample port are closed.
Store the flow cell at 4 °C for subsequent use.