Nanopore amplicon sequencing

Yoshiyuki Matsuo

May 25, 2022

Version 4

Nanopore amplicon sequencing V.4

DOI

dx.doi.org/10.17504/protocols.io.8epv5zrodv1b/v4

Yoshiyuki Matsuo¹

¹Kansai Medical University

Yoshiyuki Matsuo

DOI: dx.doi.org/10.17504/protocols.io.8epv5zrodv1b/v4

External link: https://doi.org/10.1186/s12920-022-01218-8

Protocol Citation: Yoshiyuki Matsuo 2022. Nanopore amplicon sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5zrodv1b/v4Version 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 19, 2022

Last Modified: May 25, 2022

Protocol Integer ID: 62894

Keywords: amplicon sequencing, long read, nanopore, MinION

Abstract

The two-step PCR method allows us to perform nanopore amplicon sequencing with a user-defined inner primer set combined with barcoded outer primers provided by Oxford Nanopore Technologies, taking advantage of rapid adapter attachment chemistry. This method can be applied to a wide range of sequence-based analyses, including microbiome profiling and the identification of genetic variations in targeted loci.

Materials

Reagents/Kits
KAPA2G Robust HotStart ReadyMix (2X) (Kapa Biosystems, KK5701)

PCR-grade water

PCR Barcoding Kit (Oxford Nanopore Technologies, SQK-PBK004)

E-Gel EX Agarose Gels, 1% (Thermo Fisher Scientific, G402021)

E-Gel 1 Kb Plus Express DNA Ladder (Thermo Fisher Scientific, 10488091)

Agencourt AMPure XP (Beckman Coulter, A63880)

Freshly prepared 70% ethanol

TN buffer: 10 mM Tris-HCl pH 8.0, 50 mM NaCl

QuantiFluor ONE dsDNA System (Promega, E4871)

Flow cell R9.4.1 (Oxford Nanopore Technologies, FLO-MIN106D)

Flow Cell Priming Kit (Oxford Nanopore Technologies, EXP-FLP002)

Flow Cell Wash Kit (Oxford Nanopore Technologies, EXP-WSH004)

Workflow

Two-step PCR approach for nanopore amplicon library preparation.
In the first PCR reaction, the region of interest is amplified using specific primers flanked by anchor sequences. The anchor sequences allow for a second round of PCR with Oxford Nanopore's barcoded outer primers with rapid adapter attachment chemistry. The second PCR with reduced cycle numbers generates barcoded amplicons with modified 5' ends for simplified post-PCR adapter attachment.

1st PCR with inner primers

Prepare the PCR master mix.
ABC
ComponentVolumeFinal conc.
Template DNAx µL
10 µM FW/RV primer mix0.5 µL0.2 µM each
2X KAPA2G Robust HS ReadyMix12.5 µL1X
Water12 - x µL
Total25 µL

Inner 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 outer primers used in the 2nd PCR.

Note
The following inner 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 AGRGTTYGATYMTGGCTCAG-3'

1492R:
      5'-ACTTGCCTGTCGCTCTATCTTC CGGYTACCTTGTTACGACTT-3'

Perform PCR.
ABCD
StepTemperatureTimeCycles
Initial denaturation95°C3 min1
Denaturation95°C15 sec25-35
Annealing55°C15 sec
Extension72°C30 sec
Hold4°C∞1
The above is an example for amplifying the near-full length (V1–V9) sequence of bacterial 16S rRNA genes (approximately 1,500 bp). 

Equipment
Veriti 96-Well Thermal Cycler
NAME
Applied Biosystems
BRAND
4375786
SKU

Analyze Amount2 µL   of the PCR products by gel electrophoresis to verify successful amplification.

Equipment
E-Gel Power Snap Electrophoresis Device
NAME
Thermo Fisher Scientific
BRAND
G8100
SKU

Equipment
E-Gel Power Snap Camera
NAME
Thermo Fisher Scientific
BRAND
G8200
SKU

2nd PCR with barcoded outer primers

Prepare the PCR master mix.
AB
ComponentVolume
1st PCR products1.0 µL
BP01–120.5 µL
2X KAPA2G Robust HS ReadyMix12.5 µL
Water11 µL
Total25 µL
BP01–12: barcoded outer primers supplied in the PCR Barcoding Kit.

Note
The 1st PCR products may need to be purified using AMPure XP beads before the second round of PCR. This additional step removes reaction contaminants, including primer dimers, which would be beneficial for downstream analysis.

Perform PCR.
ABCD
StepTemperatureTimeCycles
Initial denaturation95°C3 min1
Denaturation95°C15 sec8–10
Annealing62°C15 sec
Extension72°C30 sec
Hold4°C∞1
The above is an example for barcoding bacterial 16S rRNA gene amplicons (approximately 1600 bp).

Equipment
Veriti 96-Well Thermal Cycler
NAME
Applied Biosystems
BRAND
4375786
SKU

Analyze Amount1 µL   of the PCR products by gel electrophoresis.

Equipment
E-Gel Power Snap Electrophoresis Device
NAME
Thermo Fisher Scientific
BRAND
G8100
SKU

Equipment
E-Gel Power Snap Camera
NAME
Thermo Fisher Scientific
BRAND
G8200
SKU

PCR cleanup

Resuspend the AMPure XP beads by vortexing.

Add AMPure XP beads to the sample and mix by pipetting.
AB
ComponentVolume
2nd PCR products24 µL
AMPure XP12 µL

Note
To select DNA fragments of over 500 bp, add 0.5 µL AMPure XP per 1 µL of sample (0.5x ratio).

Incubate at TemperatureRoom temperature   for Duration00:05:00  .

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

Pipette off the supernatant.

Wash the beads with 70% ethanol as follows (1/2).

Keeping on the magnetic rack, add Amount200 µL   of 70% ethanol without disturbing the bead pellet.

Discard the supernatant.

Wash the beads with 70% ethanol as follows (2/2).

Keeping on the magnetic rack, add Amount200 µL   of 70% ethanol without disturbing the bead pellet.

Discard the supernatant.

Spin down and place the tube back in the magnetic rack.

Pipette off any residual ethanol.

Remove the tube from the magnetic rack and resuspend the beads in Amount10 µL   of TN buffer.
Note
TN buffer: 10 mM Tris-HCl pH 8.0, 50 mM NaCl

Incubate at TemperatureRoom temperature   for Duration00:02:00  .

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

Transfer the eluate to a new tube.

[Optional] Analyze Amount1 µL   of the purified sample by gel electrophoresis to confirm the recovery. 

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.

Equipment
Quantus Fluorometer
NAME
Promega
BRAND
E6150
SKU

Sequencing library preparation

Pool all barcoded amplicons to a total of Amount50-100 fmoles   in Amount10 µL   of TN buffer.
ABC
ComponentVolumeDNA
Sample #01 (25 ng/µL)1.0 µL25 ng
Sample #02 (25 ng/µL)1.0 µL25 ng
Sample #03 (25 ng/µL)1.0 µL25 ng
Sample #04 (25 ng/µL)1.0 µL25 ng
TN buffer6.0 µL-
Total10 µL100 ng
In the above example, four barcoded 16S rRNA gene amplicons (~1600 bp) are pooled together in equal proportions.

Note
For full-length 16S rRNA gene amplicons (approximately 1,600 bp), 50–100 fmoles of dsDNA equates to ~50–100 ng.

Add Amount1 µL   of Rapid Adapter (RAP) and mix gently by pipetting.
Note
RAP is supplied in the PCR Barcoding Kit.

Incubate at TemperatureRoom temperature   for Duration00:05:00  .

Store the library TemperatureOn ice   until ready to load.

Flow cell check

Open the MinION lid and insert the flow cell under the clip.

Equipment
MinION Mk1C
NAME
Oxford Nanopore Technologies
BRAND
M1CBasicSP
SKU

Perform flow cell check.

Check the number of active pores available for the experiment.

Sample loading

Prepare flow cell priming mix and vortex thoroughly.
AB
ComponentVolume
Flush Tether (FLT)30 µL
Flush Buffer (FB)1.17 mL
Total1.2 mL
FLT and FB are supplied in the Flow Cell Priming Kit. FB is provided in tubes, pre-aliquoted with 1.17 mL.

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 tip into the priming port.

Turn the wheel of the pipette slowly to increase the volume and draw back Amount20-30 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 Amount800 µL   of the priming mix (from Step 33) into the flow cell via the priming port.
Note
Use P1000 micropipette. Avoid introducing air.

Wait for Duration00:05:00  .

Prepare the sequencing library for loading.
AB
Component Volume
Library (from Step 29)11 µl
Water4.5 µl
Sequencing Buffer (SQB)34 µl
Loading Beads (LB)25.5 µl
Total75 µl
SQB and LB are supplied in the PCR Barcoding Kit.

Note
Mix the LB suspension well before adding it to the loading mixture.

Lift the SpotON sample port cover of the flow cell.

Load Amount200 µL   of the priming mix (from Step 33) into the flow cell via the priming port (caution: not the SpotON sample port).
Note
Use P1000 micropipette. Avoid introducing air.

Gently mix the sequencing library (Amount75 µL  , prepared in Step 38) by pipetting just prior to loading.

Load the library into the flow cell via the SpotON sample port in a dropwise fashion.
Note
Use P100 or P200 micropipette. Let each drop flow into the port before adding the next one.

Replace the SpotON sample port cover and close the priming port.

Nanopore sequencing

Start sequencing run.
AB
ParameterSetting
Flow cell typeFLO-MIN106 (R9.4.1)
KitPCR Barcoding Kit SQK-PBK004
BasecallingOn
Basecalling configurationFast basecalling
BarcodingOn
Trim barcodesOn
Barcode both endsOff
Mid-read barcode filteringOn
Q score filtering8 (default value)
Typical examples of run parameters with real-time basecalling on the MinION Mk1C.

Flushing a flow cell

Stop sequencing run.

Prepare flow cell wash mix and gently mix by pipetting.
AB
ComponentVolume
Wash Mix (WMX)2 µL
Wash Diluent (DIL)398 µL
Total400 µL
WMX and DIL are supplied in the Flow Cell Wash Kit. WMX contains DNase I.

Remove fluid in the waste channel via the waste port.
Note
Ensure that both the priming port and SpotON sample port are closed.
Use P1000 micropipette.

Open the priming port cover of the flow cell.

[Optional] If necessary, remove air bubbles under the cover by following the procedure in Step 35.

Load Amount400 µL   of the wash mix (from Step 46) into the flow cell via the priming port.
Note
Use P1000 micropipette. Avoid introducing air.

Close the priming port.

Wait for Duration01:00:00    to digest remaining DNA on the flow cell.

Remove fluid in the waste channel via the waste port.
Note
Ensure that both the priming port and SpotON sample port are closed.
Use P1000 micropipette.

Open the priming port cover of the flow cell.

[Optional] If necessary, remove air bubbles under the cover by following the procedure in Step 35.

Load Amount500 µL   of Storage Buffer (S) into the flow cell via the priming port.
Note
Buffer S is supplied in the Flow Cell Wash Kit. 
Use P1000 micropipette. Avoid introducing air.

Close the priming port.

Remove fluid in the waste channel via the waste port.
Note
Ensure that both the priming port and SpotON sample port are closed.
Use P1000 micropipette.

Store the flow cell at Temperature4 °C   for subsequent use.

A	B	C
Component	Volume	Final conc.
Template DNA	x µL
10 µM FW/RV primer mix	0.5 µL	0.2 µM each
2X KAPA2G Robust HS ReadyMix	12.5 µL	1X
Water	12 - x µL
Total	25 µL

	A	B
	Primer	Sequence
	Forward (FW)	5'-TTTCTGTTGGTGCTGATATTGC - target-specific sequence -3'
	Reverse (RV)	5'-ACTTGCCTGTCGCTCTATCTTC - target-specific sequence -3'

A	B	C	D
Step	Temperature	Time	Cycles
Initial denaturation	95°C	3 min	1
Denaturation	95°C	15 sec	25-35
Annealing	55°C	15 sec
Extension	72°C	30 sec
Hold	4°C	∞	1

	A	B
	Component	Volume
	1st PCR products	1.0 µL
	BP01–12	0.5 µL
	2X KAPA2G Robust HS ReadyMix	12.5 µL
	Water	11 µL
	Total	25 µL

A	B	C
Component	Volume	DNA
Sample #01 (25 ng/µL)	1.0 µL	25 ng
Sample #02 (25 ng/µL)	1.0 µL	25 ng
Sample #03 (25 ng/µL)	1.0 µL	25 ng
Sample #04 (25 ng/µL)	1.0 µL	25 ng
TN buffer	6.0 µL	-
Total	10 µL	100 ng

	A	B
	Component	Volume
	Flush Tether (FLT)	30 µL
	Flush Buffer (FB)	1.17 mL
	Total	1.2 mL

	A	B
	Component	Volume
	Library (from Step 29)	11 µl
	Water	4.5 µl
	Sequencing Buffer (SQB)	34 µl
	Loading Beads (LB)	25.5 µl
	Total	75 µl

	A	B
	Parameter	Setting
	Flow cell type	FLO-MIN106 (R9.4.1)
	Kit	PCR Barcoding Kit SQK-PBK004
	Basecalling	On
	Basecalling configuration	Fast basecalling
	Barcoding	On
	Trim barcodes	On
	Barcode both ends	Off
	Mid-read barcode filtering	On
	Q score filtering	8 (default value)

	A	B
	Component	Volume
	Wash Mix (WMX)	2 µL
	Wash Diluent (DIL)	398 µL
	Total	400 µL

Public workspaceNanopore amplicon sequencing V.4

Nanopore amplicon sequencing V.4