Oct 23, 2025

Metagenomic Library preparation optimized for adaptive sampling Nanopore

This  protocol  is a draft, published without a DOI.
  • 1Station Biologique de Roscoff;
  • 2CNRS-Sorbonne Université
Lucie Caradec
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Protocol Citation: Lucie Caradec, Coralie Rousseau, Gwenn Tanguy 2025. Metagenomic Library preparation optimized for adaptive sampling Nanopore. protocols.io https://dx.doi.org/
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
Created: February 12, 2025
Last Modified: October 23, 2025
Protocol  Integer ID: 119959
Keywords: nanopore, adaptive sampling, hmw, metagenome, adaptive sampling with the nanopore mk1c sequencer, nanopore mk1c sequencer, library preparation with nanopore ligation protocol, sampling nanopore presentation, nanopore, nanopore ligation protocol, oxford nanopore platform, library preparation of metagenomic sample, metagenomic library preparation, nanopore presentation, potential of nanopore, metagenomic sample, dna high molecular weight size selection, optimized library preparation, nucleotide sequence, readfish, library preparation, reference sequence database, dna
Abstract
Presentation
Adaptive sampling, also called "Readfish", was developed for the Oxford Nanopore platform. This innovative method allows the enrichment or depletion of reads of interest during real-time sequencing. The measurable ionic current, called the "squiggle", is translated into a nucleotide sequence as it passes through the nanopore and is directly compared to a reference sequence database. Depending on whether the signal aligns with a reference, the channel can briefly reverse the voltage across the pore to eject an unwanted read, thereby allowing the pore to be reused for other molecules.
Recommendations
While the use of adaptive sampling is straightforward, library preparation is one of the crucial steps to fully exploit the potential of Nanopore sequencing. In this context, this protocol results from an optimized library preparation of metagenomic samples for adaptive sampling with the Nanopore MK1C sequencer.
The protocol includes several important steps:  
1) DNA High Molecular Weight size selection to cut <10Kb
2) Quality Control
3) Library preparation with nanopore ligation protocol
4) Long Read sequencing with adaptive sampling
The reference sequence database must not excess 125 Mb.
Materials
Reagents AMPure XP beads (Agencourt) Ethanol (70%, freshly prepared) Nuclease-free water (ThermoFisher, AM9937)
DNA clean-up and size selection: Short Read Eliminator XS Kit-PacBio(102-208-200)
Qubit dsDNA BR Assay kit (Thermofisher Q32850)
Qubit dsDNA HS Assay kit (Thermofisher Q32854)
Qubit Assay tubes (Thermofisher Q32856)
Nanopore Ligation Sequencing kit (SQK-LSK114)
NEBNext FFPE Repair Mix (NEB, M6630)
NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
NEBNext Quick Ligation Module (NEB, E6056)
R10.4.1 flow cells (FLO-MIN114).
Materials 1.5 mL DNA LoBind Eppendorf tube Wide Bore Pipette Tips

Equipment
Manual pipette- P1000- P100- P10-P3 and filters tips
Magnetic rack for Eppendorf tubes
Centrifuge for 1.5mL (up to 10 000 rcf) Centrifuge for 50 mL Falcon tubes (up to 16 000 rcf) Thermomixer incubator
Hula mixer, (gentle rotator mixer) Thermofisher
LabChip GX Touch Nucleic Acid Analyzer (Revity) QuBit fluorometer (Thermofisher)

Nanopore MK1 C MinION or equivalent
Equipments
- LabChip GX Touch (or equivalent for QC check)
- MinION MK1C device
- MinION Flow Cell Light Shield
- HulaMixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Microcentrifuge
- Vortex mixer
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Timer
- Qubit fluorometer (or equivalent for QC check)



Note
Please note that the timings are incorrect : they are quite difficult to estimate and manipulator-dependent.

Materials and Consumables
Oxford Nanopore Library preparation
- 1 μg (or 100-200 fmol) high molecular weight genomic DNA
- Metagenome-Assembled-Genome sequence in fasta file (< 125 Mb)
- Oxford Nanopore Technologies Ligation Sequencing Kit V14 (SQK-LSK114)
- MinION Flow Cell (FLO-PRO114M)
- Qubit dsDNA High Sensitivity Assay kit (Invitrogen, Q32851)
- NEBNext FFPE Repair Mix (NEB, M6630)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Freshly prepared 80% ethanol in nuclease-free water
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Qubit Assay Tubes (Invitrogen, Q32856)
Size selection
- Short Read Eliminator XS Kit, PacBio
- Freshly prepared 70% ethanol in nuclease-free water
Size selection using the Short Read Eliminator XS Kit
2h 9m
Size selection removes high molecular weight DNA below 10 kb. Beforehand, a nucleic acid size check is performed on a Perkin Elmer labchip GX Touch nucleic acid analyzer with Genomic assay user guide
Figure 1. Labchip GXT profile before size selection

Adjust the DNA sample to total volume of 60 µL and a Qubit DNA concentration of 25-150 ng/µL. Pipette the sample into a 1.5ml Eppendorf DNA LoBind tube.

2m
Add 60 µL of Buffer SRE XS and mix thoroughly but gently by pipetting up and down using a wide-bore tip.

3m
Place the tube in the centrifuge and note the orientation of the tube within the rotor. Centrifuge the sample at 10.000 x g, Room temperature for 00:30:00 .
Aspirate and discard the supernatent, taking care not to disturb the pellet.
Note : the pellet may not be visible, but it will be located on the side of the tube that was facing outwards during centrifugation.

Figure 2. Picture from the guide overview Short Read Eliminator SRE XS and XL kits (https://www.pacb.com/wp-content/uploads/Guide-overview-Short-Read-Eliminator-SRE-XS-and-XL-kits.pdf)

30m
Add 200 µL of 70% ethanol to the tube, without disturbing the pellet.

1m
Centrifuge the sample at 10.000 x g, Room temperature for00:03:00 .
Discard the supernatent.

3m
Repeat steps 5.4 and 5.5.
Add50-100 µL of Buffer EB to the DNA pellet, taking in consideration the volume needed for the multiple quality controls.
Mix thoroughly but gently by pipetting up and down using a wide-bore tip.
Place the tube on the HulaMixer and leave it Overnight .
Place the tube on the thermomixer 1400 rpm, 50°C for 01:00:00 .

Note
- The pellet might still be visible. If so, repeat the next step : HulaMixer Overnight and thermomixer 1400 rpm, 50°C 00:30:00
- After that, it is important to proceed with the manipulations even if the pellet is still visible.

1h 30m
Quantify 2 times using the Qubit 1X dsDNA High Sensitivity Assay kit (or the Broad Range kit), ensuring that replicate Qubit measurements are consistent before continuing to the library preparation.
It is also recommended to validate the elimination of the short fragments by using a nucleic acid analyzer.
In this case, a nucleic acid size check was performed on a Perkin Elmer labchip GX Touch nucleic acid analyzer with Genomic assay user guide

Figure 3. LabChip GX Touch profile after size selection using Short Eliminator XS Kit

DNA repair and end-prep
1h 10m 30s
Place all the necessary reagents on ice to thaw and the Agencourt AMPure XP beads out at Room temperature .
Prepare the following in a 0.2 mL thin-walled PCR tube:
  • 47 µL DNA (input 2.5 µg, this might be over 47 μL but that is fine. Adjust the amount of beads to match the total volume of this mixture (sample + buffers/enzymes)
  • 3.5 µL NEBNext FFPE DNA Repair Buffer (vortex and spin down)
  • 3.5 µL Ultra II End-Prep Reaction Buffer (vortex and spin down)
  • 3 µL Ultra II End-Prep Enzyme Mix (do not vortex, spin down)
  • 2 µL NEBNext FFPE DNA Repair Mix (do not vortex, spin down)
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Using a thermal cycler, incubate at 20 °C for 00:30:00 and 65 °C for 00:30:00

1h
Resuspend the AMPure XP beads by vortexing.
Transfer DNA sample to a clean 1.5 mL Eppendorf DNA LoBind tube.
Add 60 µL (or equivalent volume, see step 6.1) of resuspended beads to the reaction and mix by flicking the tube.

Incubate on a hula mixer for 00:05:00 at Room temperature .

5m
Prepare 500 µL of fresh 80% ethanol in nuclease-free water.

Spin down and pellet sample on magnet until eluate is clear and colorless, about 00:02:00 .
Keep the tube on the magnet and pipette off the supernatant.

Note : You can keep the supernatant in a separated tube to make sure not to lose your sample. If the following quantification is very low, it means that the High Molecular Weight DNA is in the supernatent.
2m
Keep the tube on the magnet and wash the beads with 200 µL of 80% ethanol, pipetting on the opposite wall making sure not to disturb the pellet. Remove and discard the ethanol.

Repeat the previous step 6.10.
Spin down and place the tube back on the magnet, pipetting off any residual ethanol.
Allow to dry for ~00:00:30 but do not over-dry to the point of cracking.

30s
Remove the tube from the magnetic rack and resuspend the pellet in 61 µL nuclease-free water. Incubate for00:02:00 at Room temperature .

2m
Pellet the beads on the magnetic rack until the eluate is clear and colourless, for at least 00:01:00 .

1m
Quantify 1 µL of eluted sample on the Qubit, with the dsDNA High Sensitivity kit.

At this step, it is possible to store the sample at 4 °C overnight if needed.

Adapter ligation and clean-up
2h 35m 30s
Spin down the Ligation Adapter (LA) and NEBNext Quick T4 DNA Ligase, then return to ice.
Thaw Ligation Buffer (LNB) at Room temperature , mix by pipetting up and down and place On ice .

Thaw Elution Buffer (EB) and Short Fragment Buffer (SFB) at RT, mix by vortexing, spin down, and place on ice.
In a 1.5 mL Eppendorf DNA LoBind tube, mix the following in order:
(Between each addition, pipette mix 10-20 times)
- 60 µL DNA sample from the previous step
- 25 µL LNB
- 10 µL NEBNext Quick T4 DNA Ligase
-5 µL Ligation adapter

Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate the reaction for 00:30:00 atRoom temperature .

30m
Resuspend AMPure beads by vortexing.
Add 40 µL of resuspended beads to the reaction and mix by flicking.

Incubate on a hula mixer for 00:05:00 atRoom temperature .

5m
Spin down sample and pellet on magnet. Keeping tube on magnet, pipette off the supernatant when clear and colourless.
Note : You can keep the supernatant in a separated tube to make sure not to lose your sample. If the following quantification is very low, it means that the DNA is in the supernatent.
Wash the beads with 250 µL LFB, remove from magnet and flick to resuspend, spin down and repellet on magnet, and then remove and discard supernatant.

Repeat step 4.11.
Spin down and pellet on magnet, pipette off any residual supernatent. Allow to dry for ~00:00:30 , but do not dry the pellet to the point of cracking.

30s
Remove the tube from the magnet and resuspend the pellet in 15 µL EB.


Spin down and incubate for 02:00:00 at 37 °C .

2h
Pellet the beads on magnet until eluate is clear and colorless.
Remove and retain 15 µL of eluate containing the DNA library into a clean 1.5ml Eppendorf DNA LoBind tube.

Quantify 1 µL of eluted sample on the Qubit.

The reccomended amount of DNA is 35-50 fmol. You can use the NEB calculator (https://nebiocalculator.neb.com/#!/dsdnaamt) to estimate the amount you have.
In the case the concentration is lower than 35 fmol, you may sequence all the library.

Store the library at 4 °C until ready to load.

Priming and Loading R10 Flow Cell
5m
Note: This kit is only compatible with R10.4.1 flow cells (FLO-PRO114M).

Thaw Sequencing Buffer (SB), Library Solution (LIS) or Library Beads (LIB), Flow Cell Tether (FCT), and Flow Cell Flush (FCF) atRoom temperature , vortex, and spin down.

Open the MinION, device lid and slide the flow cell under the clip. Press firmly on the flow cell to ensure correct thermal and electrical contact. If not done before, do a flow cell check to estimate the number of pores available.
In a 1.5mL tube, add 30 µL of thawed and mixed FCT to 1170 µL of thawed and mixed FCF and vortex. This is your priming mix.

Slide the flow cell priming port cover clockwise to open the priming port.
Set a P1000 pipette to 200 µL insert the tip into the priming port and use the dial to draw back a small amount of volume to remove any air bubbles (usually about 20-30 µL , just until a small volume of buffer enters the pipette tip).

Load 800 µL of the priming mix into the flow cell via the priming port avoiding the introduction of air bubbles.

Wait for 00:05:00 .

5m
Prepare the library mix for loading :
  • 37.5 µL SB
  • 25.5 µL LIS
  • 12 µL DNA library


Complete the flow cell priming :
  • Gently lift the Spot ON port cover to make the SpotON sample port accessible.
  • Load 200 µL of the priming mix (step 6.2) into the cell priming port (NOT THE SPOTON) avoding the introduction of air bubbles.

Mix the prepared library gently by pipetting up and down just prior to loading.
Add 75 µL of prepared library to the flow cell via the SpotON sample port drop by drop. Ensure each drop flows into the port before adding the next.

Gently replace the SpotON cover on.
Install the light shield on the flowcell.

On the MK1C MinION select:
- Sequence -> then indicate the name of your experiment
- Protocol : SQK-LSK114
- Enrich or deplete, select the sequence of interest previously imported in /internal/storage/file (.fasta)
- Basecalling off
- Start Run

Protocol references
Coralie Rousseau, Sylvie Rousvoal, Catherine Leblanc, Simon M. Dittami . Microbial DNA extraction from the brown seaweed Ascophyllum nodosum. protocols.io https://protocols.io/view/microbial-dna-extraction-from-the-brown-seaweed-as-dzdr7256

M. Loose, S. Malla, M. Stout, Real-time selective sequencing using nanopore technology, Nat. Methods 13 (2016) 751–754. https://doi.org/10.1038/nmeth.3930.

A. Payne, N. Holmes, T. Clarke, R. Munro, B.J. Debebe, M. Loose, Readfish enables targeted nanopore sequencing of gigabase-sized genomes, Nat. Biotechnol. 39 (2021) 442–450. https://doi.org/10.1038/s41587-020-00746-x.

A. Payne, N. Holmes, T. Clarke, R. Munro, B. Debebe, M. Loose, Nanopore adaptive sequencing for mixed samples, whole exome capture and targeted panels, 2020. https://doi.org/10.1101/2020.02.03.926956.

S. Martin, D. Heavens, Y. Lan, S. Horsfield, M.D. Clark, R.M. Leggett, Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples, Genome Biol. 23 (2022) 11. https://doi.org/10.1186/s13059-021-02582-x.





Acknowledgements
Olivier Godfroy
Simon Dittami