May 28, 2026
Rapid Amplicon ONT Sequencing
- 1Assiut University
- Khaled Abdrabo: Yin-Tse Huang
Protocol Citation: Khaled Abdrabo 2026. Rapid Amplicon ONT Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwm3b2lmk/v1
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: April 28, 2025
Last Modified: May 28, 2026
Protocol Integer ID: 192014
Keywords: Amplicon, Oxford Nanopore Technology, Sequencing, rapid sequencing of amplicon dna, amplicon dna, rapid amplicon ont, rapid sequencing, amplicon, final sequencing, pcr amplification, dna extraction, steps from dna extraction
Abstract
This protocol enables rapid sequencing of amplicon DNA ranging from 500 bp to 5 kb. It supports sequencing of up to 96 amplicons and includes steps from DNA extraction and PCR amplification using two barcoding stages, to final sequencing.
Safety warnings
DO NOT VORTEX THE ENZYME SOLUTIONS
DNA extraction
Extract your DNA following this protocol.
1st time PCR amplification
Amplify your target amplicon sequence using the appropriate set of primer pairs following this protocol, section "For 1' PCR head-primers"
Clean-up the 1st time PCR product
After checking the success of 1st time PCR reaction by visualizing onto gel electrophorsis, proceed to clean-up step. This step is critical to ensures the removal of proteins, salts, dNTPs, and primers. To do so, follow this protocol: dx.doi.org/10.17504/protocols.io.bp2l695eklqe/v1
2nd time PCR amplification (Barcoding step)
Perform the barcoding step following the section of "For 2' PCR barcoded-head primers" in this protocol:
Check the success of the barcoding step for your amplicon through running the gel electrophorsis
Pooling
30m
After barcoding step, mix your amplicon samples together.
To avoid losing the amplicons with different lengths, follow the following principle of the pooling ratio;
Amount of long amplicon = Length ratio
Since length ratio = length of the long amplicon
End-prep
1h 30m
Note
DO NOT VORTEX THE ENZYME SOLUTIONS
Note
Mix the chemicals by flicking the tube with your finger. Spin down the tube.
Note
Place all chemicals in particular enzymes in ICE BATH
Note
It is advisable to quantify the DNA concentration after pooling step to make sure it is >100 ng/ul
In 200 µL tube(s), combine the following chemicals per tube:
| Reagents | Volume | |
| Pooled DNA | 20 ul | |
| KAPA buffer | 2.8 ul | |
| KAPA enzyme | 1.2 ul |
Incubate in a thermocycler at 20 °C for 00:30:00 and 65 °C for 00:30:00 . Hold at 4 °C
Ligation
12h
In a clean 200 µL tube, add the reagents in the following order:
Note
Ensure the Ligation Buffer is thoroughly vortexing until the droplets or any precipitations were dissolved.
| Reagents | Volume | |
| End-prep product | 24 ul | |
| Ligation Buffer (LNB) | 10 ul | |
| KAPA Ligase | 4 ul | |
| Ligation Adaptor | 2 ul |
Note
Ensure the reaction is thoroughly mixed by flicking the tube with your finger. Spin down the tube brieftly.
Incubate Overnight at Room temperature
Clean-up
30m
Thaw either Long Fragment Buffer (LFB) for over 3K amplicons or Short Fragment Buffer (SFB) for less than 3K amplicons at room temperature on a cooling block, mix by vortexing, spin down and place on ice.
Resuspend the AMPure XP beads by vortexing.
Add 0.55X AMPure XP Beads (AXP) to the tube and mix by flicking the tube.
Note
Please always determine the input DNA length on an agarose gel and then calculate the appropriate amount of AMPure beads to use.
Incubate for 00:05:00 at Room temperature
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
Wash the beads by adding either 125 µL LFB or SFB. Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet
Remove the supernatant using a pipette and discard.
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for 5~30 seconds.
Note
Do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet in 15 µL Elution Buffer (EB). Spin down and incubate for 00:10:00 37 °C On incubator
10m
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Transfer the elute into new clean tube.
Quantify the DNA concenration using Qubit.
Note
If too low (e.g. < 1 uL/ng), then stop here and start all over again.
It is recommended to be > 50 ng/ul for good library!
Note
Store the library on ice until ready to load.
Priming & loading the SpotON flow cell
Thaw the Flow cell, Sequencing Buffer (SB), Library Bead (LIB) at room temperature before mixing by vortexing.
Combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), mix by pipetting at room temperature.
| Reagent | Volume | |
| Flow Cell Flush (FCF) | 1,170 ul | |
| Flow Cell Tether (FCT) | 30 ul |
Slide the flow cell priming port cover clockwise to open the priming port.
Note
Make sure there is no air bubbles in the flow cell.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
1. Set a P1000 pipette to 800 μl.
2. Insert the tip into the priming port.
3. Turn the wheel until the dial shows 820–830 µl, or until you can see a small volume of buffer entering the pipette tip. This is a critical step, MAKE SURE NO AIR BUBBLES MOVING IN THE FLOW PATH.
Note
If happen to see air bubble in the flow path, close the priming port and try to withdraw all fluids in the waste channel from the waste port 2. Then proceed to next step.
Load 800 μl of the priming mix into the flow cell via the priming port by turning the pipet wheel, avoiding the introduction of air bubbles. Wait for 00:10:00 . During this time, prepare the library for loading by following the steps below.
Note
It is recommend to add first drop of primming solution without touching the port to watch it if it is flows into the port or settle on outside. This will help you to discover if it is still air bubbles discontinue the fluids in the flow path.
During waiting time prepare the library for loading in a new 1.5 ml Eppendorf tube as follows:
| Reagent | Volume | |
| Sequencing Buffer (SB) | 37.5 ul | |
| Library Beads (LIB) | 25.5 ul | |
| DNA library | 12 ul |
Note
The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
Complete the flow cell priming
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
Load 200 μl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
By this end all available pores on the flow cell are open!
Note
Load the library as soon as possible after this step.
Gently flipping the prepared library just prior to loading.
Note
Use 200 ul tip pipette
Keep the priming port cover open.
Add the library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
Gently close the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port, cover the sensor array with the sensory cover, and close the MinION device lid. Start the sequencing process using MinKnow software.
MinKNOW sequencing
Navigate to the start page and click Start sequencing.
Fill in your experiment details, such as name and flow cell position and sample ID.
Select the Ligation sequencing Kit (SQK-LSK114). Don't choose any sub-choice options, then directly continue.
Toggle off the Basecalling option.
Now the sequencing process will start!!
Flow Cell Wash & Storage
Stop or pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
Note
If you are not going to wash it immediately. Keep in 4°C.
Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.
Thaw one tube of Wash Diluent (DIL) at room temperature on a cooling block.
Note
Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, spin down briefly and place on ice.
In a clean 1.5 mL microtube, prepare the following Flow Cell Wash Mix:
| Reagent | Volume | |
| Wash Mix (WMX) | 1 ul | |
| Wash Diluent (DIL) | 199 ul |
Note
Mix well by pipetting, and place on ice. Do not vortex the tube.
Lift the sensory cover.
Note
Ensure that the priming port cover and SpotON sample port cover are in their positions.
Rotate the flow cell priming port cover clockwise so that the priming port is visible.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
Set a P1000 pipette to 800 μl.
Insert the tip into the priming port.
Turn the wheel until the dial shows 820–830 μl, or until you can see a small volume of liquid entering the pipette tip.
Note
Visually check that there is continuous buffer from the priming port across the sensor array.
Load 200 μl of the prepared Flow Cell Wash Mix into the flow cell via the priming port, avoiding the introduction of air.
Close the priming port and keep into 4 °C for Overnight
to #40 and redo again!
Take out the MinION device from 4°C fridge.
Thaw one tube of Storage Buffer (S) at room temperature on a cooling block.
Mix contents thoroughly by pipetting and spin down briefly.
Rotate the flow cell priming port cover clockwise so that the priming port is visible. Open the device lid.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
Set a P1000 pipette to 800 μl.
Insert the tip into the priming port.
Turn the wheel until the dial shows 820–830 μl, or until you can see a small volume of liquid entering the pipette tip.
Note
Visually check that there is continuous buffer from the priming port across the sensor array.
Add 500 μl of Storage Buffer (S) through the priming port of the flow cell by turning the pipet wheel.
Close the priming port.
Using a P1000, remove all fluids from the waste channel through Waste port 2. As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.
Note
It is vital that the flow cell priming port and SpotON sample port are closed to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
Perform a Flow Cell Check. And store it at 4°C.