Apr 23, 2025
Version 3
Nanopore Library Preparation for R10 Native Barcoding Kit V.3
- 1Kaohsiung Medical University
Protocol Citation: Yin-Tse Huang 2025. Nanopore Library Preparation for R10 Native Barcoding Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wpm2vo5/v3Version created by Yin-Tse Huang
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: March 05, 2025
Last Modified: April 23, 2025
Protocol Integer ID: 138495
Abstract
Nanopore R10 flow cell Native Barcoding library construction.
Step1. End-prep
Step1. End-prep
1h
1h
Make sure you have high concentration of DNA (>100 ng/µL, quantified by Qubit).
Use the "Volume_original" by default
In 200 µL tube(s), combine the following chemicals per tube:
| A | B | C | |
| Reagent | Volume_down | Volume_original | |
| DNA | 6.4 uL | 11.5 uL | |
| KAPA HyperPrep End Repair & A-tailing Buffer | 0.77 uL | 1.38 uL | |
| KAPA HyperPrep End Repair & A-tailing Enzyme | 0.33 uL | 0.59 uL | |
| TOTAL | 7.5 uL | 13.47 uL |
Note
Make sure the E&A buffer does not contain white precipitates before aspirate it.
Note
Mix the chemicals by flicking the tube with your finger. Spin down the tube.
Incubate in a thermocycler at 20 °C for 00:30:00 and 65 °C for 00:30:00 . Hold at 4 °C
1h
Step3. Native barcode ligation
Step3. Native barcode ligation
Thaw the Native Barcodes (NB) required for your number of samples at room temperature on a cooling block.
Note
Individually mix the barodes by vortexing, spin down, and place them on ice.
In a clean 200 µL tube, add the reagents in the following order per tube:
| A | B | C | |
| Reagent | Volume_down | Volume_original | |
| End-prep Product | 7.5 µL | 13.47 uL | |
| Native Barcode | 2.5 µL | 4.49 uL | |
| ddH2O | 1.1 µL | 1.97 uL | |
| Ligation Buffer | 6.6 µL | 11.85 uL | |
| Ligase | 2.2 µL | 3.95 uL | |
| TOTAL | 19.9 uL | 35.73 uL |
Note
Ensure the reaction is thoroughly mixed by flicking the tube with your finger. Spin down the tube brieftly.
Incubate for 20 minutes at Room temperature .
Thaw EDTA at Room temperature on a cooling block, mix by vortexing, spin down, and place on ice.
Add 2 µL (Volume_down) or 3.5 uL (Volume_original) of EDTA to each tube and mix thoroughly by flicking the tube and spin down briefly.
Note
This step is to terminate the end-repair process
Pool all the barcoded samples in a 1.5 mL microtube.
Note
Per sample, it contains
22 uL for Volume_down
39.23 for Volume_original
Prepare fresh 80% ethanol in ddH2O.
Resuspend the AMPure XP beads by vortexing.
Add 0.45X AMPure XP Beads (AXP) to the tube and mix by flicking the tube.
| A | B | C | D | E | F | G | |
| Volume_down | Volume_original | ||||||
| Number of sample | 2 | 3 | 4 | 2 | 3 | 4 | |
| AMPure | 19.8 uL | 29.7 uL | 39.6 uL | 35.3 uL | 52.9 uL | 70.6 uL | |
Incubate for 10 minutes at Room temperature .
Spin down the sample and pellet on a magnet for 5 mintues.
Keep the tube on the magnet until the elute is clear and colorless, and pipette off the supernatant.
Keep the tube on the magnetic rack and wash with 700 µL of freshly prepared 80% ethanol without disturbing the pellet.
Flip the magnetic rack for 30 seconds. Remove the ethanol using a pipet and discard.
Repeat previous step .
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 35 µL ddH2O by gently flicking.
Incubate for 10 minutes at 37 °C on an incubator .
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain 35 µL of elute into a clean 200 uL microtube.
Qubit to quatify the resulting DNA concentration before Adaptor ligation
Note
If too low (e.g. < 10 uL/ng), then stop here and start all over again
Step2. Adapter ligation
Step2. Adapter ligation
30m
30m
Thaw the Native Adapter (LA) and KAPA HyperPrep Ligation Buffer. Vortex and spin down the tubes, and place on ice immediately afterwards.
Note
Although the recommended 3rd party ligase is supplied with its own buffer, the ligation efficiency of Adapter Mix (AMX) is higher when using Ligation Buffer supplied within the Ligation Sequencing Kit.
Note
Ensure the Ligation Buffer is thoroughly vortexing until the droplets or any precipitations were dissolved.
Spin down the KAPA HyperPrep Ligase and place on ice immediately.
In a 1.5 mL tube, add the following chemicals:
| Reagent | Volume | |
| Pool Barcoded Sample | 30 µL | |
| Native Adapter (NA) | 5 µL | |
| ddH2O | 1.67 µL | |
| Ligation Buffer | 9.99 µL | |
| Ligase | 3.33 µL | |
| TOTAL | 49.99 uL |
Note
Mix the chemicals by flicking the tube with your finger. Spin down the tube.
Incubate overnight at Room temperature
Note
in ONT manual, it says at least 20 min, but it can be as longer as overnight, which may improve the adaptor ligation so the sequencing efficiency
Step3. Cleanup
Step3. Cleanup
30m
30m
Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at room temperature on a cooling block, mix by vortexing, spin down and place on ice.
Resuspend the AMPure XP beads by vortexing.
Add 0.45X AMPure XP Beads (AXP) to the tube and mix by flicking the tube.
Note
49.99 uL * 0.45X = 22.5 uL AMPure XP Beads (AXP)
Incubate for 10 minutes at Room temperature .
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
Note
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.
Wash the beads by adding either 125 µL Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet.
Note
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.
Remove the supernatant using a pipette and discard.
Repeat previous step .
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 10 minutes at 37 °C on an incubator .
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Leave the tube aside, and go to the priming steps.
Note
If you are not going to sequence the prepared library in an hour later, remove and retain 14 µl of eluate containing the DNA library into a clean 1.5 mL microtubes.
For short-term storage or reloading flow cells between washes, you can keep the prepared libray at 4°C. While for long-sterm storage of more than 3 months, storing libraries at -80°C is recommended.
Step3. Cleanup
Step3. Cleanup
30m
30m
Qubit to quatify the resulting DNA concentration before Adaptor ligation
Note
If too low (e.g. < 1 uL/ng), then stop here and start all over again
Step4. Priming and loading the SpotON flow cell
Step4. Priming and loading the SpotON flow cell
Thaw the Flow cell, Sequencing Buffer (SB), Loading Solution (LIS) or Library Bead (LIB, if using) at room
temperature before mixing by vortexing
Combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), mix by pipetting at room temperature.
| A | B | |
| Reagent | Volum per flow cell | |
| Flow Cell Flush (FCF) | 1,170 µL | |
| Flow Cell Tether (FCT) | 30 µL | |
| (OPTIONAL) Bovine Serum Albumin (BSA) at 50 mg/ml | 5 µL | |
| TOTAL | 1200 uL |
Priming and loading your flow cell
Note
Watching the priming and loading flow cell video if unsure about the process
Open the MinION device lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.
Note
Make sure there is no air ibubbles in the flow cell.
Slide the flow cell priming port cover clockwise to open the priming port.
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.
Note
Visually check that there is continuous buffer from the priming port across the sensor array.
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 5 minutes. During this time, prepare the library for loading by following the steps below.
Complete the flow cell priming:
1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
2. 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.
Note
Load the library as soon as possible after this step.
In the eluted original DNA library tube, add Sequencing Buffer (SB) and Library Solution (LIS) or Library Bead (LIB, if using):
| A | B | |
| Sequencing Buffer (SB) | 37.5 µL | |
| Library Solution (LIS) or Library Bead (LIB, if using) | 25.5 µL | |
| DNA library | 12 µL | |
| TOTAL | 75 µL |
Gently flipping the prepared library just prior to loading.
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.
Step5. Flow Cell Wash and Storage
Step5. Flow Cell Wash and Storage
30m
30m
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 µL | |
| Wash Diluent (DIL) | 199 µL |
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 the positions indicated in the figure below.
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:
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 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 2 days.
go to step #48 Repeat this step.
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:
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 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.
The flow cell can now be stored at 4°C.