Mar 16, 2026

NinjaSeq Protocol for ONT Library Preparation 

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl8xr9rv2w/v1
  • 1Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania;
  • 2Department of DNA data storage, Genomika, Kaunas, Lithuania;
  • 3Computer Science, Technion-Israel Institute of Technology, Haifa, Israel;
  • 4Computer Science, Reichman University, Herzliya, Israel
Kornelija Kaminskaitė
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl8xr9rv2w/v1
Protocol Citation: Ignas Galminas, Omer Sabary, Hadas Abraham, Kornelija Kaminskaitė, Tomer Cohen, Vakarė Gruodytė, Gediminas Alzbutas, Zohar Yakhini, Rūta Palepšienė, Lukas Žemaitis, Eitan Yaakobi, Simonas Juzėnas 2026. NinjaSeq Protocol for ONT Library Preparation . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8xr9rv2w/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: December 16, 2025
Last Modified: March 16, 2026
Protocol  Integer ID: 235104
Keywords: Restriction endonucleases, Restriction recognition sites, Sequencing, Oxford Nanopore, DNA Data Storage, ONT, Synthetic DNA, ONT library preparation, Nanopore sequencing, designated restriction enzyme, sequencing library preparation, restriction enzyme, using t4 dna ligase, t4 dna ligase, sequencing adapter, stranded dna break, dna fragment, resulting dna fragment, dna sequence, dna, dna break, ninjaseq, use with oxford nanopore technology, ont library preparation, termini of the dna sequence, conventional library preparation method, oxford nanopore technology, pcr with primer, using pcr, usage of the fd bsegi re, efficiency comparable to conventional library preparation method
Abstract
This protocol outlines the restriction enzymes (RE)-based protocol used in NinjaSeq, a novel approach to sequencing library preparation. NinjaSeq was designed and tested for use with Oxford Nanopore Technologies (ONT) sequencing and has been shown to achieve efficiency comparable to conventional library preparation methods, while reducing both cost and procedural complexity.
In NinjaSeq, DNA is first amplified using PCR with primers that contain a restriction enzyme-specific recognition sequence (RRS). This ensures that the RE recognition site is incorporated into both termini of the DNA sequence. Next, the designated restriction enzyme recognizes these sites and introduces double-stranded DNA breaks, generating overhangs compatible with sequencing adapters. The resulting DNA fragments are then ligated to adapters using T4 DNA ligase, making them ready for sequencing.
This protocol describes the usage of the FD BseGI RE. When using different REs, reaction conditions must be modified accordingly.
Materials
  1. 0.2 mL PCR tube
  2. 1.5 mL RNase- and DNase-free low-bind tube
  3. Milli-Q water (Thermo Fisher Scientific)
  4. Fast Digest Buffer (FD0293, Thermo Fisher Scientific)
  5. Fast Digest BseGI (FD0293, Thermo Fisher Scientific)
  6. AMPure XP Beads (AXP)
  7. 80% ethanol
  8. Monarch Spin PCR & DNA Cleanup Kit (5 μg) (T1130S, NEB) (optional)
  9. Ligation Buffer (LNB, ONT)
  10. Quick T4 DNA Ligase (E6056S, NEB)
  11. Ligation Adapters (LA, ONT)
  12. Small Fragment Buffer (SFB, ONT)
  13. Elution Buffer (EB, ONT)
  14. Qubit HS dsDNA kit (Thermo Fisher Scientific)
  15. ONT flow cell
  16. ONT device
  17. Flow Cell Flush (FCF, ONT)
  18. Flow Cell Tether (FCT, ONT)
  19. Sequencing Buffer (SB, ONT)
  20. Library Beads (LIB, ONT)
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 restriction enzyme and Quick T4 DNA Ligase.
  • Perform all centrifugation steps at room temperature.

Note
"ONT" refers to reagents from the standard SQK-LSK114 kit provided by Oxford Nanopore Technologies.

DNA End Preparation
10m
Transfer 100-200 ng of the purified dsDNA previously amplified with primers containing RRS on their 5’ end to a clean 0.2 mL PCR tube.
Add the following components sequentially, mixing by pipetting approximately 10 times after each ddition:
ComponentVolume
Purified dsDNAX µL (100-200 ng)
Nuclease-free water X µL (to 27 µL)
Fast Digest Buffer (FD0293, Thermo Fisher Scientific)2 µL
Fast Digest BseGI (FD0293, Thermo Fisher Scientific)1 µL
Total volume:30 µL
Briefly centrifuge the tube and place it into a thermal cycler with a heated lid.
Run the following thermal cycling program:
37 °C 00:05:00
80 °C 00:05:00 (optional)
10m
Purification
Pick the desired protocol: Purification can be performed using AMPure XP Beads or Monarch PCR & DNA Cleanup Kit.
AMPure XP Beads purification
10m 30s
Resuspend the AMPure XP Beads (AXP) thoroughly by vortexing.
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 54 µL of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.

Incubate on Hula mixer or mix by inverting by hand for 00:05:00 at Room temperature .
5m
Prepare 500 µL of fresh 80% ethanol in Nuclease-free water.

Spin down the sample and pellet on a magnet until the supernatant is clear and colorless (around 00:02:00 ). Keep the tube on the magnet, and pipette off the supernatant.

2m
Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat this step.
Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.

30s
Remove the tube from the magnetic rack and resuspend the pellet in 21 µL Nuclease-free water. Incubate for 00:02:00 at Room temperature .

2m
Pellet the beads on a magnet until the eluate is clear and colorless, for at least 00:01:00 .

1m
After incubation, transfer the reaction mixture to a 1.5 mL RNase-and DNase-free low-bind tube.
Measure DNA concentration using the Qubit HS dsDNA kit (Thermo Fisher Scientific).
Monarch® PCR & DNA Cleanup Kit approach
4m
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 70 µL Nuclease-free water into the same tube.

Thoroughly mix with pippeting.
Add 500 µL of Binding buffer, mix by pipetting.

Insert the Monarch Spin Column into the Monarch Spin Collection Tube and load the sample onto the column. Spin for 00:01:00 12000 rpm , then discard the flow-through.

1m
Re-insert the column into the collection tube. Wash by adding 200 µL of Washing buffer and spin 12000 rpm for 00:01:00 .

1m
Repeat wash.
Transfer the column to a clean 1.5 ml microfuge tube.
Add 21 µL of elution buffer to the center of the matrix to elute DNA. Wait for 00:01:00 , and spin 12000 rpm for 00:01:00 .

2m
Discard the column and measure DNA concentration using the Qubit HS dsDNA kit (Thermo Fisher Scientific).
Adapter ligation
20m
After purification, transfer 250 fmol of 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:
ComponentVolume
Purified dsDNAX µL (250 fmol)
Nuclease-free waterX µL (to 60 µL)
Ligation Adapters (LA, ONT)5 µL
Ligation Buffer (LNB, ONT)25 µL
Quick T4 DNA Ligase (E6056S, NEB)10 µL
Total volume:100 µL
Incubate the reaction at Room temperature for 00:20:00 .

20m
Purification an elution
12m 30s
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 180 µL of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.

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

5m
Spin down the sample and pellet on a magnet (around 00:02:00 ). Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.

2m
Wash the beads by adding 250 µL of 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.

Repeat the previous step.
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.

30s
Remove the tube from the magnetic rack and resuspend the pellet in 12 µL Elution Buffer (EB).

Spin down and incubate for 00:05:00 at Room temperature .

5m
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Remove and retain 12 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube and place On ice .

Measure DNA concentration using the Qubit HS dsDNA kit (Thermo Fisher Scientific).
Flow Cell Loading and Sequencing: Flow Cell Priming
Insert flow cell into a device and perform pore scan.
Prepare the flow cell priming mix by combining the following components in a 1.5 mL tube, then immediately place on ice:
ComponentVolume (Flongle)Volume (MinION)Volume (PromethION)
Flow Cell Flush (FCF, ONT)117 µL1170 µL1170 µL
Flow Cell Tether (FCT, ONT)3 µL30 µL30 µL
Total volume:120 µL1200 µL1200 µL
Carefully load the priming mix into the flow cell, ensuring no air bubbles are introduced.
Flow Cell Loading and Sequencing: Library Preparation for Sequencing
Prepare the sequencing mix by combining the following components.
ComponentVolume (Flongle)Volume (MinION)Volume (PromethION)
Sequencing Buffer (SB, ONT)15 µL37.5 µL100 µL
Library Beads (LIB, ONT)10 µL25.5 µL68 µL
Prepared LibraryX µL (50 fmol)X µL (100 fmol)X µL (100 fmol)
Elution Buffer (EB, ONT)X µLX µLX µL
Total volume:35 µL75 µL200 µL
Incubate at room temperature for 10 minutes before initiating sequencing run.