Feb 13, 2026
Modified library preparation with ligation sequencing kit DNA V14 (SQK-LSK114) from Oxford Nanopore
- laura.aguilera 1,2
- 1Centro Nacional de Análisis Genómico (CNAG), Baldiri Reixac 4, Barcelona, 08028, Spain;
- 2Universitat de Barcelona (UB), Barcelona, 08028, Spain
- Biodiversity Genomics Europe
Protocol Citation: laura.aguilera 2026. Modified library preparation with ligation sequencing kit DNA V14 (SQK-LSK114) from Oxford Nanopore. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l21dnxg1y/v1
Manuscript citation:
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: December 03, 2025
Last Modified: February 13, 2026
Protocol Integer ID: 234119
Keywords: HMW-DNA, Biodiversity Genomics Europe, BGE, short read eliminator, sre, ont library, oxford nanopore, nanopore, SQK-LSK114, long read library, long read library prep, library preparation procedure for oxford nanopore, sequencing kit dna v14, oxford nanopore this protocol, kit dna v14, library preparation with ligation, dna input, dna sample, library preparation procedure, promethion device, promethion flow cell, modified library preparation, sequencing output
Funders Acknowledgements:
Biodiversity Genomics Europe receives funding from the European Union's Horizon Europe Research and Innovation Action
Grant ID: 101059492
Disclaimer
The duration times presented are estimations only.
Abstract
This protocol describes the library preparation procedure for Oxford Nanopore sequencing of DNA samples on a Promethion device.
The DNA input and incubation conditions have been modified from the standard ONT protocol to obtain enough library to load and wash one Promethion Flow Cell 2-3 times to maximize sequencing output.
The library is expected to have an N50 around 30kb.
Attachments
Materials
6 µg of HMW DNA before size selection; or 3-4 µg HMW DNA into library prep
Consumables
- Short read eliminator (PacBio 102-208-300)
- Optional: Short Read Eliminator XS (PacBio 102-208-200)
- SQK-LSK114 or SQK-LSK114-XL (Oxford Nanopore)
- Promethion R10.4 Flow Cell (Oxford Nanopore)
- NEBNext® Companion Module v2 (NEB, E7672S or E7672L)
- Qubit dsDNA Assay Kit (Invitrogen, Q32851)
- AMPure XP Beads (AXP)
- Nuclease-free water
- Freshly prepared 70% and 80% ethanol in nuclease-free water
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml tube strips if using a thermal cycler
Equipment
- Promethion device
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- 200 μL Wide Bore Pipette Tips
- Centrifuge Eppendorf 5425 or similar
- Microfuge
- Vortex mixer
- Thermal cycler or Heat Block
- Magnetic separation rack
- Ice bucket with ice
- Qubit™ fluorometer (or equivalent for QC check)
Troubleshooting
Safety warnings
The operator must wear a lab coat, powder-free nitrile gloves to perform the laboratory procedures in this protocol.
Waste needs to be collected in a suitable container and disposed of in accordance with local regulations.
Liquid waste needs to be collected in a suitable container and disposed of in accordance with local regulations.
See best practices, thawing and handling instructions, and safety instructions for all the kit reagents, as well as instructions and safety data sheets for other reagents used in this protocol before starting.
Before start
Short read eliminator (SRE) progressively depletes fragments up to 25 kb from genomic DNA samples. If
your sample contains a lot of short fragments, you can perform SRE XS instead to avoid massive loss of the sample (depletes up to 10kb fragments).
Size selection
16h 47m
Prepare 6 µg of HMW DNA in nuclease-free water, EB buffer or TE.
High levels of salts will affect the size selection.
Mix thoroughly the SRE Buffer by vortexing.
Add 60 µl of SRE Buffer to each 60 µl of DNA. Mix well by flicking the tube and spin.
1m
Centrifuge at 10 000 x g for 00:30:00 at room temperature, with the hinge of the tube facing outwards.
30m
Immediately remove the supernatant carefully not to dislodge the pellet.
- The pellet might not be visible.
- Avoid disturbing the pellet by pipetting on the opposite side of the tube.
- The protocol allows to leave 10 µl of supernatant behind.
- Save the supernatant to repeat the procedure in case a low recovery is expected.
1m
Wash the pellet, without resuspending, with 500 µl of freshly prepared 70 % EtOH (1.5 ml tubes).
2m
Centrifuge 10,000 x g for 00:02:00 and discard the supernatant.
2m
Repeat steps 6 and 7.
Pipette off any residual ethanol.
Resuspend in 30-50 µl of Low TE buffer.
1m
If low recovery is expected, repeat the same procedure (steps 6-8) with the supernatant saved in step 5.
Allow to resuspend overnight at room temperature or at 4ºC.
- If the pellet is not fully resuspended, incubate at 50ºC or allow for resuspension at room temperature several days.
16h
Quantify 1 µl of eluted sample using a Qubit fluorometer.
Required
Concentration [ng/µl]Yield [ng]
10m
Store at 4ºC.
DNA repair and end-prep
3h 40m
Prepare the NEB reagents in accordance with manufacturer’s instructions and place on ice.
- Thaw all reagents on ice.
- Flick and/or invert the reagent tubes to ensure they are well mixed.
Note
Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.
- Always spin down tubes before opening for the first time each day.
- Vortex the FFPE DNA Repair Buffer v2 to ensure it is well mixed.
Note
The FFPE DNA Repair Buffer v2 buffer may contain a white precipitate. If this occurs, allow the mixture to come to room temperature and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix.
- The FFPE DNA Repair Buffer v2 may have a yellow tinge and is fine to use if yellow.
30m
Prepare the DNA in nuclease-free water:
- Transfer 2-3 µg input DNA into a 1.5 ml Eppendorf DNA LoBind tube. Use wide bore tips.
- Adjust the volume to 48 µl with nuclease-free water.
- Mix thoroughly by flicking the tube.
- Spin down briefly in a microfuge.
1m
In a 0.2 ml thin-walled PCR tube, mix the following:
| Reagent | Volume | |
| DNA from the previous step | 47 µl | |
| DNA CS (optional) | 1 µl | |
| NEBNext FFPE DNA Repair Buffer v2 | 7 µl | |
| NEBNext FFPE DNA Repair Mix | 2 µl | |
| Ultra II End-prep Enzyme Mix | 3 µl | |
| Total volume | 60 µl |
Reagents for DNA repair and end-prep mix
1m
Mix the reaction by flicking the tube and briefly spin down.
Using a thermal cycler, incubate at 20ºC for 00:30:00 and 65ºC for 00:30:00 . Then cool down to room temperature. Alternatively, a heat block and 1.5 ml Eppendorf DNA LoBind tubes can be used.
1h 5m
Resuspend the AMPure XP Beads (AXP) by vortexing.
1m
Spin down and transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
CleanUp 1x:
Add 60 µl of resuspended AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
1m
Incubate for00:15:00 at room temperature.
15m
Freshly prepare 1 ml of 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
5m
Keep the tube on the magnet and wash the beads with 500 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
1m
Repeat the previous step.
1m
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
2m
Remove the tube from the magnetic rack and resuspend the pellet in 61 µl nuclease-free water by flicking the tube.
1m
Incubate for 01:30:00 at 37ºC.
1h 30m
Spin down the tube and pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
5m
Remove and retain 60 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Use wide bore tips.
1m
The sample can progress to adapter ligation or be stored at 4ºC overnight.
Adapter ligation and clean-up
3h 40m
Prepare all the reagents.
- Spin down the Ligation Adapter (LA) and Salt-T4® DNA Ligase, and place on ice.
- Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
- Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.
- Thaw the Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and keep at room temperature.
20m
In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:
| Reagent | Volume | |
| DNA sample from the previous step | 60 µl | |
| Ligation Adapter (LA) | 5 µl | |
| Ligation Buffer (LNB) | 25 µl | |
| Salt-T4® DNA Ligase | 10 µl | |
| Total | 100 µl |
Reagents for the adapter ligation mix
3m
Thoroughly mix the reaction by flicking the tube and briefly spinning down.
1m
Incubate the reaction for 01:00:00 at 20ºC or room temperature.
1h
Resuspend the AMPure XP Beads (AXP) by vortexing.
Cleanup 0.4x:
Add 40 µl of resuspended the AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.
1m
Incubate for 00:15:00 at room temperature.
15m
Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet and pipette off the supernatant.
5m
Wash the beads by adding 250 µl Short Fragment Buffer (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.
4m
Repeat the previous step.
4m
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet in 31 µl nuclease-free water by flicking the tube. Incubate for 01:30:00 at 37ºC.
1h 32m
Spin down the tube and pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
5m
Remove and retain 30 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube. Use wide bore tips.
Quantify 1 µl of eluted sample using a Qubit fluorometer.
Required
Concentration [ng/µl]Yield [ng]
10m
Prepare your library in 30ul of Elution buffer (EB).
- The library prepared following this protocol is expected to be >20kb. Prepare 500-600ng of library to load on the Promethion R10.4 FC following manufacturer’s instructions. After loading, check that the pore occupancy is above 95% to optimize the output. If pore occupancy is below 94%, try to top-up by adding more library (there is space to load 200ul more in the flow cell).
- Store the remaining library at 4ºC to maximize the flow cell output by washing the flow cell and reloading fresh library when the active pores drop below 30% during the sequencing run.
Protocol references