Feb 12, 2025

Public workspaceNanopore amplicon sequencing V.5

DOI
dx.doi.org/10.17504/protocols.io.8epv5zrodv1b/v5
  • 1Kansai Medical University
Yoshiyuki Matsuo
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DOI: dx.doi.org/10.17504/protocols.io.8epv5zrodv1b/v5
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/v5Version 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: May 20, 2022
Last Modified: February 12, 2025
Protocol Integer ID: 119466
Keywords: amplicon sequencing, long read, nanopore, MinION
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 Mk1B (Oxford Nanopore Technologies)
  • MinION Flow Cell R10.4.1 (Oxford Nanopore Technologies, FLO-MIN114)
  • Apple Silicon Mac (e.g., Mac Studio with M1 Max and MacBook Pro with M1 Pro)
Protocol materials
ReagentPCR Barcoding Expansion 1–12Oxford Nanopore TechnologiesCatalog #EXP-PBC001
ReagentPCR Barcoding Expansion 1–96Oxford Nanopore TechnologiesCatalog #EXP-PBC096
ReagentProNex Size-Selective Purification SystemPromegaCatalog #NG2001
ReagentQuantiFluor ONE dsDNA SystemPromegaCatalog #E4871
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
ReagentLigation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
ReagentSalt-T4 DNA LigaseNew England BiolabsCatalog #M0467S
ReagentUltraPure™ BSA (50 mg/mL)Thermo Fisher ScientificCatalog # AM2616
ReagentFlow Cell Wash KitOxford Nanopore TechnologiesCatalog #EXP-WSH004
ReagentPrimeSTAR Max DNA Polymerase Ver.2Takara Bio Inc.Catalog #R047A
Workflow
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
First-round PCR with tailed primers
Prepare the PCR master mix.
ABC
ComponentVolumeFinal conc.
Template DNAx µL
5 µM FW/RV primer mix1.5–2.5 µL0.3–0.5 µM each
PrimeSTAR Max Ver.2 Premix (2X)12.5 µL1X
Waterup to 25 µL
ReagentPrimeSTAR Max DNA Polymerase Ver.2Takara Bio Inc.Catalog #R047A

Tailed primers (user-supplied)
AB
PrimerSequence
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 16S rRNA gene. 16S rRNA gene-specific sequences are in bold letters.

  • 27F:
5'-TTTCTGTTGGTGCTGATATTGC AGRRTTYGATYHTDGYTYAG-3'

  • 1492R:
5'-ACTTGCCTGTCGCTCTATCTTC CRGHTACCTTGTTACGACTT-3'

Perform PCR.
ABCD
StepTemperatureTimeCycles
Denaturation98°C10 sec25–35
Annealing57°C15 sec
Extension68°C5–10 sec/kb*
Hold4°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.
Second-round PCR with barcoded primers
Second-round PCR with barcoded primers
Prepare the PCR master mix.
AB
ComponentVolume
First-round PCR products1.0 µL
PCR Barcode (BC)*0.5 µL
PrimeSTAR Max Ver.2 Premix (2X)12.5 µL
Water11 µL
Total25 µL
*One of barcoded primers (10 µM BC) supplied in the PCR Barcoding Expansion (EXP-PBC001 or EXP-PBC096).
ReagentPCR Barcoding Expansion 1–12Oxford Nanopore TechnologiesCatalog #EXP-PBC001
ReagentPCR Barcoding Expansion 1–96Oxford Nanopore TechnologiesCatalog #EXP-PBC096

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, including primer dimers, which would be beneficial for the downstream analysis.

Perform PCR to incorporate barcode sequences.
ABCD
StepTemperatureTimeCycles
Denaturation98°C10 sec5–10
Annealing57°C15 sec
Extension68°C5–10 sec/kb*
Hold4°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
PCR clean-up
14m
14m
Allow the ProNex beads to equilibrate to room temperature.

ReagentProNex Size-Selective Purification SystemPromegaCatalog #NG2001
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. The samples can be pooled in desired ratios, and the total volume can be flexible, depending on the experimental requirements.

Alternatively, each sample can be cleaned up separately prior to pooling. After quantifying the DNA, the samples can be pooled in the desired ratios and subsequently subjected to the End-prep (step 28).

Add ProNex beads to the pooled sample and mix by pipetting.
AB
ComponentVolume
Pooled sample100 µL
ProNex Chemistry100 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 TemperatureRoom temperature for Duration00:05:00 .

5m
Place the tube on a magnetic rack for Duration00:02:00 .
Equipment
NGS MagnaStand v.3 8Ch
NAME
Magnetic rack (0.2 mL tube)
TYPE
FastGene
BRAND
FG-SSMAG3
SKU

2m
Pipette off the supernatant.
Wash the beads with 80% ethanol as follows:

Keeping on the magnetic rack, add Amount200 µL of 80% ethanol without disturbing the bead pellet.
Discard the supernatant.
Repeat steps 14.1 and 14.2 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 Amount26 µL of water.
Incubate at TemperatureRoom temperature for Duration00:05:00 .

5m
Place the tube on the magnetic rack for Duration00:02:00 .

2m
Transfer the eluate to a new tube.

DNA quantification
DNA quantification
Warm QuantiFluor ONE dsDNA dye to TemperatureRoom temperature .

ReagentQuantiFluor ONE dsDNA SystemPromegaCatalog #E4871
Add Amount1 µL of eluted sample to Amount200 µL of QuantiFluor ONE dsDNA dye in 0.5 mL tube.

Mix thoroughly by vortexing.
Incubate at TemperatureRoom temperature for Duration00:05:00 , protected from light.

5m
Measure fluorescence using the Quantus Fluorometer to quantify DNA concentration.
Equipment
Quantus Fluorometer
NAME
Promega
BRAND
E6150
SKU

End-prep
End-prep
Prepare 200 fmol of pooled DNA (from step 22) in Amount25 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.
AB
ComponentVolume
Pooled amplicon DNA25 µL
NEBNext Ultra II End Prep Reaction Buffer3.5 µL
NEBNext Ultra II End Prep Enzyme Mix1.5 µL
Total30 µL
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
Incubate the reaction using a thermal cycler.
AB
TemperatureTime
20°C5 min
65°C5 min
4°C∞

Note
Amplicons are converted into 5ʹ-phosphorylated and 3ʹ-dA-tailed DNA, enabling ligation of Nanopore sequencing adapters.


Clean-up
Clean-up
14m
14m
Allow the ProNex beads to equilibrate to room temperature.
Add ProNex beads to the end-prep reaction and mix by pipetting.
AB
ComponentVolume
End-prepped DNA30 µL
ProNex Chemistry30 µL

Note
The ratio of beads to sample volume should be adjusted accordingly. As an alternative to ProNex beads, AMPure XP can also be used.

Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Place the tube on a magnetic rack for Duration00:02:00 .
2m
Pipette off the supernatant.
Wash the beads with 80% ethanol as follows:
Keeping on the magnetic rack, add Amount200 µL of 80% ethanol without disturbing the bead pellet.
Discard the supernatant.
Repeat steps 36.1 and 36.2 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 Amount30 µL of water.
Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Place the tube on the magnetic rack for Duration00:02:00 .
2m
Transfer the eluate to a new tube.
Adapter ligation
Adapter ligation
10m
10m
Mix the following components.
AB
ComponentVolume
End-prepped DNA30 µL
Ligation Buffer (LNB)*12.5 µL
Ligation Adapter (LA)2.5 µL
Salt-T4 DNA Ligase5 µL
Total50 µL
*The Ligation Buffer (LNB) is viscous and should be thoroughly mixed by pipetting.

ReagentLigation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
ReagentSalt-T4 DNA LigaseNew England BiolabsCatalog #M0467S
Incubate at TemperatureRoom temperature for Duration00:10:00 .
10m
Clean-up
Clean-up
12m
12m
Allow the ProNex beads to equilibrate to room temperature.
Add ProNex beads to the adapter-ligated sample and mix by pipetting.
AB
ComponentVolume
Adapter-ligated DNA50 µL
ProNex Chemistry50 µL

Note
The ratio of beads to sample volume should be adjusted accordingly. As an alternative to ProNex beads, AMPure XP can also be used.

Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Place the tube on a magnetic rack for Duration00:02:00 .
Pipette off the supernatant.
Wash the beads with either Short Fragment Buffer (SFB) or Long Fragment Buffer (LFB) as follows:
Note
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 Amount100 µL of either SFB or LFB.

Place the tube on the magnetic rack for Duration00:02:00 .
2m
Discard the supernatant.
Repeat steps 52.1–52.3 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 Amount15 µL of Elution Buffer (EB).
Incubate at TemperatureRoom temperature for Duration00:05:00 .
5m
Place the tube on the magnetic rack for Duration00:02:00 .
Transfer the eluate to a new tube.
DNA quantification
DNA quantification
Warm QuantiFluor ONE dsDNA dye to TemperatureRoom temperature .

Add Amount1 µL of eluted sample to Amount200 µL of QuantiFluor ONE dsDNA dye in 0.5 mL tube.

Mix thoroughly by vortexing.
Incubate at TemperatureRoom temperature for Duration00:05:00 , protected from light.

Measure fluorescence using the Quantus Fluorometer to quantify DNA concentration.
Flow cell check
Flow cell check
Connect the MinION to the host computer and start the MinKNOW software.
Equipment
MinION
NAME
Sequencer
TYPE
Oxford Nanopore Technologies
BRAND
MinION 1B / MinION 1C
SKU

Open the MinION lid and insert a flow cell (R10.4.1) under the clip.
Equipment
MinION/GridION Flow Cell (R10.4.1)
NAME
Flow cell
TYPE
Oxford Nanopore Technologies
BRAND
FLO-MIN114
SKU
https://store.nanoporetech.com/flow-cell-r10-4-1.html
LINK

Perform flow cell check.
Check the number of active pores available for the experiment.
Remove the flow cell from the MinION device and place it in the tray.
Flow cell priming
Flow cell priming
5m
5m
Prepare the flow cell priming mix.
AB
ComponentVolume
Flow Cell Tether (FCT)30 µL
50 mg/mL Bovine Serum Albumin (BSA)5 µL
Flow Cell Flush (FCF)1,170 µL
Total1,205 µL
ReagentUltraPure™ BSA (50 mg/mL)Thermo Fisher ScientificCatalog # AM2616
Open the priming port cover of the flow cell.
Remove air bubbles under the cover as follows (if any):
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, keeping the sensor array of the flow cell covered by the buffer.

Load the priming mix into the flow cell via the priming port as follows:
Note
Avoid introducing air.

Using a P1000 micropipette, aspirate Amount800 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 Duration00:05:00 .
5m
Lift the SpotON sample port cover upwards to uncover the sample port on the flow cell.
Load the remaining Amount200 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
Sample loading
For 1–10 kbp amplicons, prepare 35–50 fmol of DNA library (from step 60) in Amount12 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.
AB
ComponentVolume
DNA library12 µL
Sequencing Buffer (SB)37.5 µL
Library Beads (LIB)*25.5 µL
Total75 µL
*Since the Library Beads (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 Amount75 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
Nanopore sequencing
Set up a sequencing run on the MinKNOW software.
AB
ParameterSetting
Flow cell typeFLO-MIN114 (R10.4.1)
KitLigation Sequencing Kit SQK-LSK114
Expansion packPCR Barcoding Expansion 1–12 EXP-PBC001
BasecallingOn
Basecalling modelFast basecalling
BarcodingOn
Trim barcodesOn
Barcode both endsOn
Q score filtering8 (default value)
Min/Max read length*Optional
Above are typical examples of run parameters for real-time basecalling on the MinION Mk1B, 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
Flow cell washing and storage for reuse
1h 5m
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.
AB
ComponentVolume
Wash Mix (WMX)2 µL
Wash Diluent (DIL)398 µL
Total400 µL
ReagentFlow 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 73.
Load the wash mix into the flow cell via the priming port as follows:
Note
Avoid introducing air.

Using a P1000 micropipette, aspirate Amount200 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 Duration00:05:00 .

5m
Load the remaining Amount200 µL of the wash mix into the priming port by following the procedure in steps 92.1–92.3.
Close the priming port.
Wait for Duration01:00:00 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 73.
Using a P1000 micropipette, load Amount500 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 Temperature4 °C for subsequent use.