Ligation-based Amplicon barcoding (Long Protocol)

Summary: This detailed protocol shows how PCR amplicons can be barcoded and prepared for Nanopore sequencing.

Protocol information

Protocol Credits

Author(s)
- Luis E. Vázquez-Quiñones, Inter American University of Puerto Rico – Arecibo Campus
- Miguel Urdaneta-Colón, University of Puerto Rico - Río Piedras Campus
- José Agosto-Rivera, University of Puerto Rico - Río Piedras Campus
Maintainer/contact: Luis E. Vázquez-Quiñones, Inter American University: email
Last updated: February, 2025
Source protocols and references
- Ligation sequencing amplicons - Native Barcoding Kit 96 V14 (SQK-NBD114.96)
- Qubit™ 1X dsDNA HS Assay

DNA source material

Type: Any double-stranded DNA
Collection source: Amplicon from PCR reaction

Nanopore Sequencing

Sequencing format: MinION OR
Sequencing kit: Native Barcoding Kit 96 V14 (SQK-NBD114.96)
Oxford Nanopore Sequencing protocol: - Ligation sequencing amplicons - Native Barcoding Kit 96 V14 (SQK-NBD114.96)
Indexed/Barcoded: Yes, 96 indicies
Samples per run: 96 samples

Computer and Bioinformatics

Analysis tools
- Varies/ not covered in this protocol
Analysis difficulty: N/A
Command line needed: N/A
GPU/Super-high accuracy basecalling required: Varies depending on application

Reagents

Personal protective equipment

As recommended by original protocols (e.g., gloves, lab coat)

Sample collection and prep

Will vary depending on application. This protocol starts with cleaned, amplified PCR product.

DNA extraction

Not applicable. User will need to provide 200 fmol (130 ng for 1 kb amplicons) DNA per sample to be barcoded.

DNA prep, library creation, and sequencing

Nanopore kit
- Ligation sequencing amplicons - Native Barcoding Kit 96 V14 (SQK-NBD114.96)
  - Native Adapter
  - Sequencing Buffer
  - Library Beads OR Library solution
  - Elution buffer
  - AMPure XP beads
  - Long Fragement Buffer OR Short Fragement Buffer
  - EDTA
  - Flow Cell Flush
  - Flow Cell Tether
  - Native Barcode Plate
Qubit™ 1X dsDNA HS Assay
- Qubit™ reagents; dsDNA HS Assay Kit (ThermoFisher® Q32851)
  - Invitrogen Qubit Assay Tubes Catalog number: Q32856 0.5 mL PCR microtubes thin-wall, clear or Axygen PCR-05-C tubes (VWR, part no. 10011-830)
  - Qubit dsDNA HS Reagent
  - Qubit dsDNA HS Buffer
  - Qubit dilution buffer
User-provided reagents
- NEB Blunt/TA Ligase Master Mix (NEB, cat No. M0367)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, cat No. E7546)
- NEBNext Quick Ligation Module (NEB, cat No. E6056)
- Agencourt AMPure XP beads
- Nuclease-free water (e.g. ThermoFisher, cat No. AM9937)
- Freshly prepared 80% ethanol in nuclease-free water (500 µL per PCR reaction to be cleaned up)
- 10 mM Tris-HCl pH 8.0 with 50 mM NaCl

Equipment and consumables

Lab equipment

Micropipette set (e.g., P10, P100, P1000) and tips
Microcentrifuge (20,000 x g)
Heat block or water bath
Qubit (dsDNA HS Assay Kit - ThermoFisher, Q32851; and assay tubes) or Nanodrop spectrophotometer for DNA quantification
Permanent markers
Magnetic rack for microfugue tubes(1)
PCR machine (for incubation of samples in PCR tubes)
Vortex mixer

Consumables

Assorted tube racks (microfuge, conical tubes, and PCR tubes)
1.5-1.7ml microfugue tubes
0.2 ml thin-walled PCR tubes
(Optional) 1.5 ml Eppendorf DNA LoBind tubes

Nanopore sequencing equipment

Sequencing device: MinION or Flongle w/Adaptor

Computer equipment

Desktop or laptop

Recommendation - Sergi Lab Supplies

Estimated timings

DNA End preperation: 30-45 mins

Stop-point: Store the samples overnight at 4 °C.

Barcode ligation: time 60-90 mins

Stop-point: Store the samples overnight at 4 °C

Adapter ligation: 60-70 mins

Stop-point: Store the samples overnight at 4 °C or long-term at -80 °C.

Flow cell loading: 20-30 mins

Background

The ONT Ligation Sequencing procedure is a method of preparing DNA molecules for sequencing in a Nanopore system using different dsDNA sources. It is a flexible method that can be used with gDNA, cDNA, or amplicons. The throughput is higher than the ONT Rapid methods but is more expensive because of reagents from other companies and has a longer time for preparation. Native Barcoding with ligation is a method for multiplex samples to reduce the cost of the samples. Multiplexing may be used only when the amount of data required is less than the total of data that can be generated from a single flow cell.

The preparation of the sample has four principal steps:

the DNA end preparation,
the Barcode ligation,
the adapter ligation and
loading the flow cell.

Additional Reading

Some reading and potentially links to references.

Library Preperation - Native Barcoding Prep (96)

Goal: Attach sequencing barcodes and adaptors necessary for nanopore sequencing.

Nanopore

This protocol follows the MinION version of Native Barcoding Kit 96 V14 (SQK-NBD114.96) from Oxford Nanopore.

Preliminary Steps

Goal: Ensure DNA ends are enzymatically prepared to attach barcodes.

End-repair

1. Add 200 fmol (130 ng for 1 kb amplicons) of each sample into a clean 96-well microplate (if you have 96 samples or prepare microtubes for the number of samples available).
2. Complete the volume of each sample up to 11.5 μL with nuclease-free water.
3. Pipette to mix gently, and spin down afterwards.
4. In each well or microtube, mix the following components.
✓ Ingredient Volume
200 fmol Amplicon DNA

11.5 µL
Diluted DNA Control Sample (DCS)

1 µL
Ultra II End-prep Reaction Buffer

1.75 μL
Ultra II End-prep Enzyme Mix

0.75 μL
5. Make sure all the ingredients are well/microtube combined by pipetting and briefly spinning them down.
6. In a thermal cycler Incubate for five minutes at 20 °C and five minutes at 65 °C.

Native Barcode Ligation

Preliminary steps

Note

Refer to the manufacturer's instructions to prepare the NEB Blunt/TA Master Mix and place it on ice.

1. Thaw all the reagents at room temperature.
2. Spin down the tubes containing the reagent for 5 seconds.
3. Perform 10 full volume pipette mixes to make sure that all reagents are thoroughly mixed.
- a. Thaw the AMpure XP Beads (AXP) at room temperature and then mix with a vortex. Then keep them at room temperature.
- b. Thaw the EDTA at room temperature and then mix with a vortex, spin down, and then place on ice.
- c. According to the number of samples to be used, select the number of Native Barcodes from NB01 ![][image4] to NB96 and thaw at room temperature.
  
  Note
  
  Individually mix the barcodes by pipetting, spin down, and then place on ice.
- d. For every individual sample select a unique barcode.
  
  Note
  
  At the final steps of the preparation, you will mix all the barcoded samples and run on the same flow cell.
- e. Using a new 96 well place add the reagents in the following order.
  ✓ Ingredient Volume
  Nuclease free water 3 µL
  End-prepared DNA 0.75 µL
  Individual Native Barcode 1.25 μL
  Blunt/TA Ligase Master Mix 5 μL

Pause point

At this step, you may stop the procedure and store the sample overnight at 4°C.

Instructions
- a. Mix the reaction thoroughly by gently pipetting and briefly spinning down.
- b. Incubate at room temperature for 20 minutes.
2. To each well or 0.2 mL microtube, add the following volume:
✓ Clear cap EDTA Blue cap EDTA
1 µL

or

2 µL
3. Then mix thoroughly by pipetting and spin down briefly.
4. Pool all individual samples and purify them with the AMPure Beads on a magnetic rack.
- a. Pool all the individual samples into one 1.5 mL Eppendorf DNA LoBind microtube.
- b. Resuspend by vortexing the AMPure XP Beads (AXP).
- c. Add 0.4X AMPure XP Beads (AXP) beads to the Pooled Reaction then mix by pipetting.
- d. Mix on a rotator mixer (Hula Mixer or other) for 10 minutes at room temperature.
- e. During the mix step prepare 2 mL of 80% ethanol in Nuclease-free water.
- f. When finished with the mix, spin down the sample and put it on a magnetic rack to pellet for 5 minutes.
  
  Note
  
  You will notice that the liquid turns clear, and the pellet is aside on the magnet.
- g. Without removing the microtube from the magnetic rack carefully remove the clear portion with a pipette without disturbing the pellet.
- h. Do not remove the tube from the magnetic rack and wash without disturbing the pellet with 700 µL of the freshly prepared 80% ethanol.
- i. With the tube on the rack carefully remove and discard the ethanol.
- j. Repeat the previous step to remove the ethanol.
- k. Take out the microtube from the rack and spin it down.
- l. Place back the tube on the magnetic rack and pipette out any alcohol.
- m. Allow the pellet to dry for approximately 30 seconds.
  
  Note
  
  Move quickly to avoid excess drying that can crack the beads.
- n. Take out the microtube from the magnetic rack and add 35 µL of Nuclease-free water.
- o. Gently flick the tube with your finger.
- p. Incubate the sample for 10 minutes at 37 °C and agitate every 2 minutes by gently flicking the tube with your finger.
- q. Put the sample back on the magnetic rack until the eluate is completely clear.
- r. Without disturbing the pellet, remove and retain the clear eluate in a new, clean 1.5 mL Eppendorf DNA LoBind microtube.
- s. DNA quantification: Quantify the sample using a Qubit® Fluorometer, Nanodrop®, QIAxpert® or other.

Pause point

At this step, you may stop the procedure and store the sample overnight at 4°C.

Adapter ligation and clean-up

Adapter ligation

Important

Do not interchange the Native Adapter (NA) of this kit with another sequencing adapter.

Prepare the NEBNext Quick Ligation Reaction Module according to the instructions of the manufacturer.

1. Thaw the following reagents at room temperature:
- a. Native Adapter (NA)
- b. Quick T4 DNA Ligase
- c. NEBNext Quick Ligation Reaction
- d. Elution Buffer (EB)
Mix by vortexing.
- e. Short Fragment Buffer (SFB) ; for all sizes DNA fragments or Long Fragments Buffer (LFB) for fragments of 3 kb or longer.

Important

The Quick T4 DNA Ligase should not be vortexed.

2. Spin down all reagents in a microcentrifuge for 5 seconds.
3. Mix thoroughly the reagents by pipetting up and down 10 times.

Important

The Quick T4 DNA Ligase should not be vortexed.

4. The Native Adapter (NA) and Quick T4 DNA Ligase must be first spin down, followed by pipette mixing and afterwards placed on ice.
5. Mix thoroughly the Elution Buffer (EB) ![A screenshot of a computerDescription automatically generated][image1] by vortexing, spin down, and place on ice.

Important

The clean-up step following adaptor ligation is intended to either enrich DNA segments of > 3 kb or purify all fragments equally, depending on the wash buffer (LFB or SFB) utilized. Use Long Fragment Buffer (LFB) to enrich DNA pieces 3 kb or longer. Use Short Fragment Buffer (SFB).

6. Mix in the following order these reagents in a 1.5 mL Eppendorf LoBind tube.
✓ Ingredient Volume
Pooled barcoded sample

30 µL
Native Adapter (NA)

5 µL
NEBNext Quick Ligation Reaction (5X)

10 µL
Quick T4 DNA Ligase

5 µL
7. Spinning down briefly.
8. Incubate for 20 minutes at Room Temperature.

Clean-up using the AMPure XP Beads

Important

This procedure uses (LFB) or (SFB) but not 80% ethanol to wash the beads because ethanol will be detrimental to the sequencing reaction. The sequencing reaction will be hampered if ethanol is used.

Important

It is recommended to load 10-20 fmol of the final prepared library onto the R10.4.1 ONT MinION flow cell (FLO-MIN114). The final prepared library should be stored on ice until ready for loading.

DNA quantification

1. Quantify the sample using a Qubit™ Fluorometer (Invitrogen™, Q32856), Nanodrop™, QIAxpert® or other for DNA quantification.

Steps to quantify the DNA

1. Set aside two microtubes for the standards (dsDNA High Sensitivity needs 2 standards) and one microtube per sample.
2. Label the microtubes on the lid (DO NOT label on the side), the instrument will be reading the sample through the microtube side.
3. The working solution needs to be prepared in a plastic tube diluting the dsDNA HS reagent 1:200 in dsDNA HS Buffer.
- a. Now use your Qubit reagent calculator and indicate the number of samples & 2 standards.
- b. Prepare the Qubit working solution by mixing the volume of Qubit dye and the volume of Qubit dilution.

Important

Do not mix the working solution in a glass container.

*Figure 1: Qubit home screen: activate the Reagent Calculator button*This image shows the Qubit home screen in which the user can choose an assay (dsDNA, RNA, Oligo, Protein, Ion Sphere, and Fluorometer). In addition, at the bottom of the screen in the middle part, the Reagent Calculator is located, and we need to activate it to compute the reagent recipe that we need to quantify our samples.

*Figure 2: Qubit Reagent Calculator*This image shows the Qubit Reagent Calculator screen: The user needs to input the number of samples in the first field, then the user inputs the number of standards in the second field. As the third step, the user needs to check the box to include overage (1 extra tube). Once the user inputs the above information, the results for the recipe appear on screen to prepare the reagent. Once the user writes down the recipe on a piece of paper, click on the Done button to return to the main screen.
(2-1) Type the number of samples, (2-2) Type the number of standards, (2-3) Select include overage, (2-4) Use the results to prepare the reagent, and (2-5) Active the Done button when finished

4. Add the working solution to the microtubes:
- Standards
  - 190 µL of the working solution per microtube
- Samples
  - 199 µL of the working solution per microtube
5. Add the samples:
- Standards
  - 10 µL of the standard to their corresponding microtube and mix it by vortexing for 2–3 seconds to avoid creating bubbles.
- Samples
  - 1 µL of the standard into their corresponding microtube and mix it by vortexing for 2–3 seconds to avoid creating bubbles.
6. Incubate the samples and standards for 2 minutes at room temperature.
7. Now select the assay type dsDNA HS (High Sensitivity; Figure 3-4).

*Figure 3: Qubit home screen: Choose an assay - dsDNA*This image shows the Qubit home screen so the user can select an assay in our case we need to select dsDNA.

*Figure 4: Qubit: choose an assay screen: dsDNA - High sensitivity*This image shows the Qubit screen so the user can select dsDNA: High sensitivity.

8. Read the standards (Figure 5)

*Figure 5: dsDNA — High sensitivity screen: Read the standards.*This image shows the Qubit screen to activate the button to read the standards.

*Figure 6: dsDNA: High sensitivity screen: Read Standard 1*This image shows the Qubit screen instructing the user to insert standard 1 and activate the read standard button and then insert standard 2 and activate the read standard button.

a. Read standard 2.
9. Now, read the samples and start preparing the pooled sample.

Example

Example: Quantifying DNA with the Qubit

Sample	[DNA] (ng/µL)
1	32.8
2	29.6
3	41.2
4	44.0
5	41.6
6	27.4

☐ Identify the sample with the highest [DNA].
☐ Divide the highest concentration by the concentration of each sample to obtain the volume that needs to be transferred for the pool.

Sample	[DNA] (ng/µL)	Volume for pooling the samples
1	32.8	44.0 / 32.8 \= 1.34 µL \= 1.3 µL
2	29.6	44.0 / 29.6 \= 1.49 µL \= 1.5 µL
3	41.2	44.0 / 41.2 \= 1.07 µL \= 1.1 µL
4	44.0	44.0 / 44.0 \= 1.00 µL \= 1.0 µL
5	41.6	44.0 / 41.6 \= 1.06 µL \= 1.1 µL
6	27.4	44.0 / 27.4 \= 1.61 µL \= 1.6 µL
	Total	7.6 µL

☐ Compute the total mass
Total DNA ng = Total volume in the pooled sample (µL) x [Highest DNA] (ng/µL)
Total DNA ng = 7.6 µL x 44.0 ng/µL = 334.4 ng

10. Connect to the NEBioCalculator and compute the fmol for your pooled samples.

Figure 7: Computing the femtomoles of DNA in our pooled sampleThis image shows the New England Biolabs Calculator to compute the double stranded DNA mass to moles:Step 1: Select ds: Mass to Moles or viceversa.Step 2:2a) Type the DNA length.2b) Select the DNA length units.Step 3:3a) Type the DNA mass.3b) Select the DNA mass units.Step 4: Now you have the fmol of DNA in your pooled sample.
- a. Select ds: Mass ⇄ Moles
- b. Type the DNA length; you need to determine the average length of the fragments to compute the fmoles. In our case, the average length for our amplicon is 1,500 bp.
- c. Select the DNA length units.
- d. Type the DNA mass.
- e. Select the DNA mass units; Now you have the fmol of DNA in your pooled sample.
11. Compute the pooled samples volume that we need to transfer to the sample for sequencing.

$\frac{12 µL}{X µL} = \frac{361.9 fmol}{20 fmol}$
$240 = 361.9 x$
$\frac{240}{361.9} = \frac{361.9 x}{361.9}$
$x = 0.66 µL$

Priming and loading the SpotON flow cell

Important

The “Ligation sequencing amplicons – Native Barcoding Kit 96 V14 (SQK-NBD114.96)” is only compatible with R10.4.1 flow cells (FLO-MIN114).

Preliminary steps

Before combining by vortexing, thaw at room temperature the following reagents spin them down once thawed and store them on ice.

✓	Reagents
	Sequencing Buffer (SB)
	One tube of Flow Cell Flush (FCF)
	Library Beads (LIB) for most experiments or Library Solution (LIS)
	Flow Cell Tether (FCT)
	50 mg/mL Bovine Serum Albumin (BSA)

Important

To get the best sequencing performance and improved output on MinION R10.4.1 flow cells (FLO-MIN114), it is recommended to add Bovine Serum Albumin (BSA) to the flow cell priming mix to a final concentration of 0.2 mg/mL. BSA ensures that more DNA strands are available for the nanopores by blocking non-specific binding in the flow cell, which boosts sequencing output.

1. Combine the following reagents to prepare the flow cell priming mix with BSA and mix by inverting the tube and pipette mix at room temperature.

Ingredient	Vol. (+ BSA)	Vol. (- BSA)
Flow Cell Flush (FCF)	1,170 µL	1,170 µL
50 mg/mL Bovine Serum Albumin (BSA)	5 µL
Nuclease-free water		5 µL
Flow Cell Tether (FCT)	30 µL	30 µL
Total	1,205 µL	1,205 µL

Note

BSA ensures that more DNA strands are available for the nanopores by blocking non-specific binding in the flow cell, which boosts sequencing output.
his solution contains a hydrophobic tether molecule used to anchor the nucleic acid onto the membrane close to the nanopore.

2. Slide the flow cell inside under the MinION’s clip by opening its lid; to ensure correct thermal and electrical contact press down firmly the flow cell.

Important

When removing the transport buffer from the flow cell, proceed with caution. Set your P-1000 micropipette to 500 µL and remove 20-30 µL of the transportation buffer slowly by only using the piston of your micropipette. Removing more than 20–30 μL is not advised. It is of the utmost importance that the buffer always covers the array of pores. Pores might sustain irreversible damage when air bubbles are introduced into the array.

Ending the experiment

1. Stop the sequencing by clicking on the stop button.
2. If you would like to reuse the flow cell after your sequencing experiment is over, please follow the instructions in the Flow Cell Wash Kit and keep the cleaned flow cell at 2-8 °C.
3. Alternatively, clean out the flow cell and prepare it for return to Oxford Nanopore by following the returns protocol.

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✓	Ingredient	Volume
	200 fmol Amplicon DNA	11.5 µL
	Diluted DNA Control Sample (DCS)	1 µL
	Ultra II End-prep Reaction Buffer	1.75 μL
	Ultra II End-prep Enzyme Mix	0.75 μL

✓	Ingredient	Volume
	Nuclease free water	3 µL
	End-prepared DNA	0.75 µL
	Individual Native Barcode	1.25 μL
	Blunt/TA Ligase Master Mix	5 μL

✓	Ingredient	Volume
	Pooled barcoded sample	30 µL
	Native Adapter (NA)	5 µL
	NEBNext Quick Ligation Reaction (5X)	10 µL
	Quick T4 DNA Ligase	5 µL