Jul 11, 2025

Public workspaceONT Library Prep for Whole Genome Sequencing

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpzp61lzp/v1
  • Jasmine Sakr1,
  • Dayeon Cheong1
  • 1UCI
Jasmine Sakr
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpzp61lzp/v1
Protocol Citation: Jasmine Sakr, Dayeon Cheong 2025. ONT Library Prep for Whole Genome Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzp61lzp/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: October 08, 2024
Last Modified: July 11, 2025
Protocol Integer ID: 109313
Keywords: Nanopore, ONT, Long read, whole genome sequencing, genome, DNA, Oxford Nanopore, Fiber-seq, ont library prep for whole genome sequencing, using oxford nanopore platform, oxford nanopore platform, preparation genomic dna for whole genome, whole genome sequencing, preparation genomic dna, genomic dna, whole genome, genome, ont library prep, sequencing, dna, library prep
Funders Acknowledgements:
NHGRI
Grant ID: HG012077
Abstract
This protocol outlines the preparation genomic DNA for whole genome sequencing using Oxford Nanopore platform.
Guidelines
Longer incubation with AMPure beads leads to higher yield.
Materials
REAGENTS

ItemSupplierPart NumberNotes
Ligation Sequencing Kit V14Oxford Nanopore TechnologiesSQK-LSK114The Short Fragment Buffer (SFB) will NOT be used because it selects for fragments that are 3kb and longer.
NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation SequencingNew England BioLabsE7180
AMPure® XP ReagentBeckman CoulterA63880 (5 mL) A63881 (60 mL)
Nuclease-Free WaterSigma-AldrichW4502Or equivalent nuclease-free water.
Ethyl Alcohol, PureSigma-Aldrich459844Or equivalent 100% non-denatured ethanol.

EQUIPMENT
ABCD
ThermomixerVarious SuppliersVariesCompatible with 1.5 mL tubes.
MicrocentrifugeVarious SuppliersVariesCompatible with 1.5 mL tubes.
Single Channel Pipettes: P2, P20, P200, P1000Various SuppliersVaries
Vortex-Genie 2Scientific IndustriesSI-0236Or an equivalent vortex mixer.
6-Tube Magnetic Separation RackNew England BiolabsS1506SOr an equivalent magnetic rack for 1.5 mL tubes.
Qubit Flex FluorometerThermo Fisher ScientificQ33327Or an equivalent fluorometer.
HulaMixer™ Sample MixerThermo Fisher Scientific15920D
SUPPLIES
ABCD
Pipette Tips TR LTS 10 μL, 20 μL, 200 μL, 1,000 μLRainin17014961 17014963 17014967 Or appropriate sterile, DNA low-binding, and filtered pipette tips. We do not recommend using wide bore tips.
DNA LoBind® Tubes, 1.5 mL, Snap CapEppendorf022431021Or equivalent DNA low-binding, nuclease-free 1.5 mL tubes.
Qubit dsDNA HS (High Sensitivity) Assay KitThermo Fisher ScientificQ33230 (100 assays) Q33231 (500 assays)Or equivalent fluorescent DNA dye based quantification kit.
Ice bucketVarious SuppliersVaries

Troubleshooting
Preparation
Prepare the following:
For each sample: Adjust Amount1.4 µg of DNA volume to Amount47 µL with Nuclease-free water in a 1.5 mL tube and mix using a wide-bore tip.

Prepare fresh 80% ethanol for the wash steps. Each sample will need 400 μL.
Set two heat blocks toTemperature65 °C and Temperature37 °C .

End-prep
End-prep Reaction
In a 1.5 mL tube, mix the following for each sample:

ItemVolume (μL)
DNA CS (optional)1
DNA sample47
Ultra II End-prep Reaction Buffer3.5
Ultra II End-prep Enzyme Mix3
NEBNext FFPE DNA Repair Buffer3.5
NEBNext FFPE DNA Repair Mix2
If you do not use DNA CS, add 1 µL of Nuclease-free water instead.

Thoroughly mix by pipetting using a wide-bore tip and avoid creating bubbles.
Incubate at TemperatureRoom temperature for Duration00:05:00 on bench top and Temperature65 °C for Duration00:05:00 on thermomixer.

10m
Clean-Up
Vortex AMPure XP Beads (AXP).
Add Amount60 µL of the resuspended AMPure XP Beads (AXP) to the end-prep reaction(s).

Incubate tube(s) on the Hula mixerShaker10 rpm, Room temperature , 00:05:00 .

Spin down the tube(s) and pellet on a magnet.
Keep the tube(s) on the magnet and pipette off the supernatant.
Keep the tube(s) on the magnet and wash the beads with Amount200 µL of freshly prepared 80% ethanol without disturbing the pellet.

Remove the ethanol using a pipette and discard without disturbing the pellet.
Keep the tube(s) on the magnet and wash the beads with Amount200 µL of freshly prepared 80% ethanol without disturbing the pellet (again).

Remove the ethanol using a pipette and discard without disturbing the pellet (again).
Allow to dry for ~30 seconds, but do NOT dry the pellet to the point of cracking.
Remove tube(s) from magnet and resuspend the pellet in Amount61 µL Nuclease-free water.

Incubate tube(s) for minimum Duration00:10:00 at Temperature37 °C .

10m
Pellet the beads on a magnet.
Remove and retain Amount60 µL of eluate into a new 1.5 mL tube(s).

Adapter Ligation
20m
Adapter ligation reaction
In the same 1.5 mL tube, mix in the following order for each sample:


ItemVolume (μL)
DNA sample (from the previous step)60
Ligation Adapter (LA)5
Ligation Buffer (LNB)25
NEBNext Quick T4 DNA Ligase10

Thoroughly mix by gently pipetting using a wide-bore tip and avoid creating bubbles.
Incubate tube(s) at TemperatureRoom temperature for Duration00:10:00 .

10m
Clean-up
Vortex AMPure XP Beads (AXP).
Add Amount40 µL of the resuspended AMPure XP Beads (AXP) to the reaction(s).

Incubate tube(s) on the Hula mixer at Shaker10 rpm, Room temperature , 00:05:00 .

Spin down the tube(s) and pellet on a magnet.
Keep the tube(s) on the magnet and pipette off the supernatant.
Remove tube(s) from magnet and wash the beads by adding Amount250 µL Short Fragment Buffer (SFB).

Resuspend the beads by pipetting with wide-bore tip.
Pellet the beads on a magnet and pipette off the supernatant.
Remove tube(s) from magnet and wash the beads by adding Amount250 µL Short Fragment Buffer (SFB) (again).

Resuspend the beads by pipetting with wide-bore tip (again).
Pellet the beads on a magnet and pipette off the supernatant (again).
Allow to dry for ~30 seconds, but do NOT dry the pellet to the point of cracking.
Remove tube(s) from magnet and resuspend the pellet in Amount32 µL Elution Buffer (EB).

Incubate for minimum Duration00:10:00 at Temperature37 °C

10m
Pellet the beads on a magnet.
Remove and retain Amount32 µL of eluate into a new 1.5 mL tube(s).

Qubit each library and keep on ice until loading or store in -20°C.