Jun 30, 2025

Public workspaceHigh-Performance Protocol for ONT Library Preparation V.2

PLOS One
Peer-reviewed method
DOI
https://dx.doi.org/10.17504/protocols.io.dm6gp973jvzp/v2
  • Lukas Žemaitis1,
  • Rūta Palepšienė1,
  • Simonas Juzėnas1,
  • Gediminas Alzbutas1,2,
  • Pierre-Yves Burgi3,
  • Thomas Heinis4,
  • Jérôme armet5,
  • Silvia Angeloni Suter5,
  • Martin Jost6,
  • Renaldas Raišutis7,
  • Friedrich Simmel8,
  • Ignas Galminas9
  • 1Department of DNA data storage, Genomika, Kaunas, Lithuania;
  • 2Institute for Digestive Research, Academy of Medicine, Lithuanian University of Health Sciences, Kaunas, Lithuania;
  • 3Department of Information and Communication Systems and Technologies, University of Geneva, Geneva, Switzerland;
  • 4Department of Computing, Imperial College London, London, United Kingdom;
  • 5School of Engineering HE-Arc Ingénierie, HES-SO University of Applied Sciences Western Switzerland, Neuchâtel, Switzerland;
  • 6MABEAL GmbH, Graz, Austria;
  • 7Ultrasound Research Institute, Kaunas University of Technology, Kaunas, Lithuania;
  • 8Department of Bioscience, TU Munich, School of Natural Sciences, Garching, Germany;
  • 9Genomika
Rūta Palepšienė
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gp973jvzp/v2
Protocol Citation: Lukas Žemaitis, Rūta Palepšienė, Simonas Juzėnas, Gediminas Alzbutas, Pierre-Yves Burgi, Thomas Heinis, Jérôme armet, Silvia Angeloni Suter, Martin Jost, Renaldas Raišutis, Friedrich Simmel, Ignas Galminas 2025. High-Performance Protocol for ONT Library Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp973jvzp/v2Version created by Rūta Palepšienė
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: June 20, 2025
Last Modified: June 30, 2025
Protocol Integer ID: 220624
Keywords: DNA end preparation, Adapter ligation, Purification and elution, Flongle Flow Cell Loading and Sequencing, Flow Cell Priming, Library Preparation for Sequencing, ligation sequencing amplicons v14, performance protocol for ont library preparation, standard ont library preparation, refined version of the oxford nanopore technology, oxford nanopore technology, ont library preparation, enhancing dna yield, dna yield, sequencing performance, sequencing, dna, ont
Funders Acknowledgements:
European Innovation Council Pathfinder Program
Grant ID: 101115389
Abstract
This protocol presents a refined version of the Oxford Nanopore Technologies (ONT) Ligation Sequencing amplicons V14 (SQK-LSK114) protocol, optimized for the sequencing of short and ultra-short DNA fragments. It maintains the use of reagents required for standard ONT library preparation while enhancing DNA yield and improving sequencing performance.

The protocol can be successfully used for all types of Flow Cells, but the Flow Cell priming and loaded library volumes must be adjusted accordingly.
Materials
  • 0.2 mL PCR tube
  • 1.5 mL RNase- and DNase-free low-bind tube
  • Milli-Q water (Thermo Fisher Scientific)
  • Ultra II End-Prep Reaction Buffer (E7546S, NEB)
  • Ultra II End-Prep Enzyme Mix (E7546S, NEB)
  • Ligation Buffer (LNB, ONT)
  • Quick T4 DNA Ligase (E6056S, NEB)
  • Ligation Adapters (LA, ONT)
  • Small Fragment Buffer (SFB, ONT)
  • Elution Buffer (EB, ONT)
  • Qubit HS dsDNA kit (Thermo Fisher Scientific)
  • Flongle (ONT) flow cell
  • MinION Mk1B device (ONT)
  • Flow Cell Flush (FCF, ONT)
  • Flow Cell Tether (FCT, ONT)
  • Sequencing Buffer (SB, ONT)
  • Library Beads (LIB, ONT)

ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
ReagentNEBNext Quick Ligation ModuleNew England BiolabsCatalog #E6056S

Troubleshooting
Before start
  • Thaw all the reagents and keep them on ice throughout the protocol.
  • Prior to use, thoroughly mix all components by vortexing, except for the End-Prep Enzyme Mix and Quick T4 DNA Ligase.
  • Perform all centrifugation steps at room temperature.
Note
Note: "ONT" refers to reagents from the standard SQK-LSK114 kit provided by Oxford Nanopore Technologies.

DNA End Preparation
10m
Transfer Concentration250 Mass Percent of the dsDNA to a clean 0.2 mL PCR tube, adjusting the volume based on DNA concentration.
Pipetting
Add the following components sequentially, mixing by pipetting approximately 10 times after each addition:
AB
ComponentVolume
dsDNAX µL (250 fmol)
Milli-Q water (Thermo Fisher Scientific)X µL (to 50 µL)
Ultra II End-Prep Reaction Buffer (E7546S, NEB)7 µL
Ultra II End-Prep Enzyme Mix (E7546S, NEB)3 µL
Total volume:60 µL
Pipetting
Mix
Briefly centrifuge the tube and place it into a thermal cycler with a heated lid.
Run the following thermal cycling program:
Temperature20 °C for Duration00:05:00 .
Temperature65 °C for Duration00:05:00 .
10m
Adapter Ligation
20m
After incubation, transfer the reaction mixture to a 1.5 mL RNase- and DNase-free low-bind tube.
Add the following components in the specified order, mixing by pipetting approximately 10 times after each addition:

AB
ComponentVolume
End-prepped DNA60 µL
Ligation Buffer (LNB, ONT)25 µL
Quick T4 DNA Ligase (E6056S, NEB)10 µL
Ligation Adapters (LA, ONT)5 µL
Total volume100 µL

Pipetting
Mix
Incubate the reaction at TemperatureRoom temperature for Duration00:20:00 .

20m
Incubation
Purification and Elution
23m
Add AMPure XP Beads (AXP, ONT) to the reaction tube based on the DNA length:
  • Amount180 µL for fragments < 100 bp.
  • Amount50 µL for fragments > 100 bp.

Pipetting
Mix by inverting the tube for Duration00:05:00 .

5m
Mix
Briefly centrifuge the tube, then place it on a magnetic rack for Duration00:03:00 to pellet the beads.

3m
Centrifigation
While keeping the tube on the magnet, carefully remove the supernatant.
Add Amount250 µL of Small Fragment Buffer (SFB, ONT) to the tube.

Pipetting
Remove the tube from the magnet and gently rotate for at least Duration00:03:00 , occasionally flicking to ensure even bead distribution.
3m
Briefly centrifuge the tube and place it back on the magnet for Duration00:03:00 , then remove the supernatant.
3m
Centrifigation
Repeat the wash step with Amount250 µL of SFB following the same procedure.

After the final wash, briefly centrifuge the tube, then place it on the magnet to remove any remaining supernatant.
Centrifigation
Allow the bead pellet to dry for Duration00:01:00 before removing the tube from the magnet.

1m
Add Amount12 µL of Elution Buffer (EB, ONT) to the tube and mix thoroughly by pipetting.

Pipetting
Mix
Incubate at TemperatureRoom temperature for Duration00:05:00 .

5m
Incubation
Place the tube back on the magnet for Duration00:03:00 to pellet the beads.

3m
Transfer the supernatant containing the prepared library to a new 1.5 mL RNase- and DNase-free tube and place TemperatureOn ice .
Pipetting
Measure DNA concentration using the Qubit HS dsDNA kit (Thermo Fisher Scientific).
Flongle Flow Cell Loading and Sequencing: Flow Cell Preparation
Insert a Flongle (ONT) flow cell into a MinION Mk1B device (ONT).
Perform a quality check using MinKNOW software.
Select only flow cells with ≥ 50 active pores for sequencing.
Flongle Flow Cell Loading and Sequencing: Flow Cell Priming
5m
Prepare the flow cell priming mix by combining the following components in a 1.5 mL tube, then immediately place TemperatureOn ice :
AB
ComponentVolume
Flow Cell Flush (FCF, ONT)117 µL
Flow Cell Tether (FCT, ONT)3 µL
Total volume:120 µL
Peel back the seal tab of the Flongle flow cell to expose the sample port.
Secure the seal tab by adhering it to the MinION lid using its adhesive strip.
Carefully load the priming mix into the flow cell, ensuring no air bubbles are introduced.
Incubate the flow cell for Duration00:05:00 .

5m
Incubation
Flongle Flow Cell Loading and Sequencing: Library Preparation for Sequencing
10m
During the incubation period, prepare the sequencing mix by combining the following components:
AB
ComponentVolume
Sequencing Buffer (SB, ONT)15 µL
Library Beads (LIB, ONT)10 µL
Prepared LibraryX µL (50 fmol)
Elution Buffer (EB, ONT)X µL
Total volume:35 µL
Incubate at TemperatureRoom temperature for Duration00:10:00 before initiating sequencing run.

10m
Incubation