Nanopore amplicon sequencing

Yoshiyuki Matsuo

Published: 2022-05-26 DOI: 10.17504/protocols.io.8epv5zrodv1b/v4

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.

Steps

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.

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

Inner 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 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.

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

The above is an example for amplifying the near-full length (V1–V9) sequence of bacterial 16S rRNA genes (approximately 1,500 bp).

Equipment

Value	Label
Veriti 96-Well Thermal Cycler	NAME
Applied Biosystems	BRAND
4375786	SKU
http://www.thermofisher.com/us/en/home.html	LINK

Analyze 2µL of the PCR products by gel electrophoresis to verify successful amplification.

Equipment

Value	Label
E-Gel Power Snap Electrophoresis Device	NAME
Thermo Fisher Scientific	BRAND
G8100	SKU
http://www.thermofisher.com/us/en/home.html	LINK

Equipment

Value	Label
E-Gel Power Snap Camera	NAME
Thermo Fisher Scientific	BRAND
G8200	SKU
http://www.thermofisher.com/us/en/home.html	LINK

2nd PCR with barcoded outer primers

Prepare the PCR master mix.

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

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.

A	B	C	D
Step	Temperature	Time	Cycles
Initial denaturation	95°C	3 min	1
Denaturation	95°C	15 sec	8–10
Annealing	62°C	15 sec
Extension	72°C	30 sec
Hold	4°C	∞	1

The above is an example for barcoding bacterial 16S rRNA gene amplicons (approximately 1600 bp).

Equipment

Value	Label
Veriti 96-Well Thermal Cycler	NAME
Applied Biosystems	BRAND
4375786	SKU
http://www.thermofisher.com/us/en/home.html	LINK

Analyze 1µL of the PCR products by gel electrophoresis.

Equipment

Value	Label
E-Gel Power Snap Electrophoresis Device	NAME
Thermo Fisher Scientific	BRAND
G8100	SKU
http://www.thermofisher.com/us/en/home.html	LINK

Equipment

Value	Label
E-Gel Power Snap Camera	NAME
Thermo Fisher Scientific	BRAND
G8200	SKU
http://www.thermofisher.com/us/en/home.html	LINK

PCR cleanup

Resuspend the AMPure XP beads by vortexing.

Add AMPure XP beads to the sample and mix by pipetting.

A	B
Component	Volume
2nd PCR products	24 µL
AMPure XP	12 µL

Note

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

10.

Incubate at Room temperature for 0h 5m 0s.

11.

Place the tube on a magnetic rack for 0h 2m 0s.

Equipment

Value	Label
NGS MagnaStand v.3 8Ch	NAME
Magnetic rack (0.2 mL tube)	TYPE
FastGene	BRAND
FG-SSMAG3	SKU

12.

Pipette off the supernatant.

13.

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

13.1.

Keeping on the magnetic rack, add 200µL of 70% ethanol without disturbing the bead pellet.

13.2.

Discard the supernatant.

14.

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

14.1.

Keeping on the magnetic rack, add 200µL of 70% ethanol without disturbing the bead pellet.

14.2.

Discard the supernatant.

15.

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

16.

Pipette off any residual ethanol.

17.

Remove the tube from the magnetic rack and resuspend the beads in 10µL of TN buffer.

Note

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

18.

Incubate at Room temperature for 0h 2m 0s.

19.

Place the tube on a magnetic rack for 0h 2m 0s .

20.

Transfer the eluate to a new tube.

20.1.

[Optional] Analyze 1µL of the purified sample by gel electrophoresis to confirm the recovery.

DNA quantification

21.

Warm QuantiFluor ONE dsDNA dye to Room temperature .

22.

Add 1µL of eluted sample to 200µL of QuantiFluor ONE dsDNA dye in 0.5 mL tube.

23.

Mix thoroughly by vortexing.

24.

Incubate at Room temperature for 0h 5m 0s, protected from light.

25.

Measure fluorescence using the Quantus Fluorometer to quantify DNA concentration.

Equipment

Value	Label
Quantus Fluorometer	NAME
Promega	BRAND
E6150	SKU
http://www.promega.com	LINK

Sequencing library preparation

26.

Pool all barcoded amplicons to a total of 50-100fmoles in 10µL of TN buffer.

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

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.

27.

Add 1µL of Rapid Adapter (RAP) and mix gently by pipetting.

Note

RAP is supplied in the PCR Barcoding Kit.

28.

Incubate at Room temperature for 0h 5m 0s.

29.

Store the library On ice until ready to load.

Flow cell check

30.

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

Equipment

Value	Label
MinION Mk1C	NAME
Oxford Nanopore Technologies	BRAND
M1CBasicSP	SKU
http://nanoporetech.com/	LINK

31.

Perform flow cell check.

32.

Check the number of active pores available for the experiment.

Sample loading

33.

Prepare flow cell priming mix and vortex thoroughly.

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

FLT and FB are supplied in the Flow Cell Priming Kit. FB is provided in tubes, pre-aliquoted with 1.17 mL.

34.

Open the priming port cover of the flow cell.

35.

Remove air bubbles under the cover as follows (if any).

35.1.

Set the volume of P1000 micropipette to 200 µL.

35.2.

Insert the tip into the priming port.

35.3.

Turn the wheel of the pipette slowly to increase the volume and draw back 20-30µL 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.

36.

Load 800µL of the priming mix (from Step 33) into the flow cell via the priming port.

Note

Use P1000 micropipette. Avoid introducing air.

37.

Wait for 0h 5m 0s.

38.

Prepare the sequencing library for loading.

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

SQB and LB are supplied in the PCR Barcoding Kit.

Note

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

39.

Lift the SpotON sample port cover of the flow cell.

40.

Load 200µL 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.

41.

Gently mix the sequencing library (75µL, prepared in Step 38) by pipetting just prior to loading.

42.

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.

43.

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

Nanopore sequencing

44.

Start sequencing run.

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)

Typical examples of run parameters with real-time basecalling on the MinION Mk1C.

Flushing a flow cell

45.

Stop sequencing run.

46.

Prepare 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 and DIL are supplied in the Flow Cell Wash Kit. WMX contains DNase I.

47.

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.

48.

Open the priming port cover of the flow cell.

49.

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

50.

Load 400µL of the wash mix (from Step 46) into the flow cell via the priming port.

Note

Use P1000 micropipette. Avoid introducing air.

51.

Close the priming port.

52.

Wait for 1h 0m 0s to digest remaining DNA on the flow cell.

53.

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.

54.

Open the priming port cover of the flow cell.

55.

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

56.

Load 500µL 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.

57.

Close the priming port.

58.

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.

59.

Store the flow cell at 4°C for subsequent use.

Nanopore amplicon sequencing

Abstract

Steps

Workflow

1st PCR with inner primers

2nd PCR with barcoded outer primers

PCR cleanup

DNA quantification

Sequencing library preparation

Flow cell check

Sample loading

Nanopore sequencing

Flushing a flow cell

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