May 27, 2026

Microbial cfDNA Library Preparation and Sequencing with Oxford Nanopore

DOI
https://dx.doi.org/10.17504/protocols.io.x54v99e84v3e/v1
  • 1Statens Serum Institut
Martin Schou Pedersen
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v99e84v3e/v1
Protocol Citation: Martin Schou Pedersen 2026. Microbial cfDNA Library Preparation and Sequencing with Oxford Nanopore. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v99e84v3e/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: May 27, 2026
Last Modified: May 27, 2026
Protocol  Integer ID: 317994
Keywords: mcfDNA, cfDNA, microbial, metagenomic, oxford nanopore, amplification-free, free dna sequencing with oxford nanopore, microbial cfdna library preparation, cfdna extraction, protocol for native barcoded cfdna, native barcoded cfdna, oxford nanopore this method, free dna sequencing, free microbial cell, department of sequencing, unbiased metagenomic amplification, sequencing
Abstract
This method is developed for unbiased metagenomic amplification-free Microbial Cell-Free DNA Sequencing with Oxford Nanopore sequencing using PromethION flow cells.
The method has been developed by Martin Schou Pedersen, Department of Sequencing and Bioinformatics, Statens Serum Institut, Copenhagen, Denmark.

This method requires cfDNA extraction prior to library prep.
This was developed before Oxford Nanopore released their protocol for Native Barcoded cfDNA so minor changes are found compared to theirs.
Materials
ABC
Native Barcoding Kit 96 V14SQK-NBD114.96Oxford Nanopore
PromethION Flow Cell R10.4.1FLO-PRO114MOxford Nanopore
Flow Cell Wash KitEXP-WSH004Oxford Nanopore
LoBind DNA 2 mL tubes525-0131VWR
Qubit dsDNA HS Assay KitQ33231Thermo Fisher
Qubit Flex Assay Tube StripsQ33252Thermo Fisher
AmpureXP beadsA63881Beckman Coulter
DynaMag-2 magnet12321DThermo Fisher
NEBNext FFPE DNA Repair v2 moduleE7360LNEB
NEBNext Ultra II End Repair / dA-tailingE7546LNEB
NEB Blunt/TA Ligase Master MixM0367LNEB
NEBNext Quick Ligation ModuleE6056LNEB
PCR foil sealing5100401CDACOS
Eppendorf Thermomixer5382000016Eppendorf
CAPPRondo High Speed MinicentrifugeCR-1512DACOS
Safety warnings
Please comply with local rules and SOP for handling the reagents and materials in this protocol.
Before start
  • Room temperature incubations require 20–25°C. Use a Thermomixer or thermocycler if the room temperatyre is outside this range.
  • Input to library prep is extracted cfDNA. Expected input: 15–30 ng in 46 µL.
  • Around 6-12 samples can be run per flow cell to obtain enough library to load. We have typically prepared more samples and split across multiple flow cells at the same time.
End Repair and dA-Tailing (End Prep)
Before starting, take out 96-well plate cold block, 2 mL tube cold block, and AmpureXP beads. Prepare all thermocycler programs beforehand.
In a PCR plate, prepare per sample:
cfDNA (15–30 ng) 46 µL
FFPE DNA Repair Buffer v2 7 µL
NEBNext FFPE DNA Repair Mix v2 2 µL.

Mix 10× by pipetting and centrifuge plate briefly
Note
If normalizing between samples, add nuclease-free H₂O to bring all samples to 46 µL input.

Run thermocycler program cfDNA-repair: 37°C for 15 min (lid 50°C) → 4°C hold.
Centrifuge plate. Place on cold block. Add 2 µL NEBNext Proteinase K directly to each well. Mix 10× and centrifuge briefly.
Run thermocycler program cfDNA-proteinaseK: 37°C for 15 min (lid 75°C) → 65°C for 5 min → 4°C hold.
Centrifuge plate. Place on cold block. Add 3 µL NEBNext Ultra II End Prep Enzyme Mix to each well. Mix 10× and centrifuge briefly.
Run thermocycler program cfDNA-end-prep: 20°C for 30 min (lid 75°C) → 65°C for 30 min → 4°C hold.

Note
NOTE: While incubating: label 2 mL LoBind tubes for next step, take out AmpureXP beads, and prepare 10 mL 80% ethanol (8 mL 100% EtOH + 2 mL water).

Remove end-prep plate from thermocycler and centrifuge briefly. Place on ice block until ready to cotinue.
Add 180 µL AmpureXP beads per sample into individual 2 mL LoBind tubes.
Add 60 µL of end-prepped DNA to the AmpureXP tube. Mix thoroughly by pipetting.
Incubate at room temperature for 5 minutes, flicking/inverting approximately every minute.
Centrifuge briefly and place on magnet until liquid is clear.
Remove supernatant. Add 200 µL 80% ethanol. Remove supernatant. Repeat wash once more.
Centrifuge briefly, return to magnet, and remove last traces of ethanol with a 10 µL tip.
Add 10 µL nuclease-free water. Gently resuspend with P10 or P50 pipette set to 7 µL.
Incubate at room temperature for 5 minutes.
Centrifuge briefly and place on magnet for 1 minute.
Transfer supernatant (containing DNA) to a new 2 mL LoBind tube.
Note
DNA can be stored at 4°C overnight before barcode ligation.

Barcode Ligation
Thaw NEB Blunt/TA Ligase Master Mix, EDTA, and Native Barcodes. Keep on ice.
Centrifuge Ligase Master Mix and barcodes briefly.
Mix Ligase Master Mix by pipetting. Add 10 µL to each 2 mL LoBind tube.
Add 7.5 µL eluted DNA to each tube.
Add 2.5 µL of a unique barcode (NB01–NB96) to each tube.
Gently mix reactions with P50/P100 pipette set to 12 µL.
Note
Record which barcode is used for each sample.

 Incubate at room temperature for 20 minutes.
Note
While incubating, set Thermomixer to 37°C.

Add 4 µL EDTA (blue cap) to each reaction and mix.
Pool all barcoded samples into one 2 mL LoBind tube. Add 3× AmpureXP volume per sample (e.g., 6 samples = 360 µL beads: (20 µL × 3) × 6).
Note
ONT's later protocols suggests only 1.2× AmpureXP. Microbial cfDNA is historically shorter than human, why we have chosen a higher AmpureXP ratio.

Incubate at room temperature for 10 minutes.
Centrifuge briefly. Place on magnet for at least 5 minutes until liquid is clear
Remove supernatant.
Add 600 µL SFB.
Wait 30 seconds.
Remove supernatant.
Add 600 µL SFB.
Wait 30 seconds.
Remove supernatant.
Centrifuge briefly and return to magnet.
Remove last traces of supernatan with a 10 µL tip.
Add 35 µL nuclease-free water. Gently resuspend with P50 pipette set to 25 µL.
 Incubate at 37°C on Thermomixer for 10 minutes, flicking tube gently for ~10 seconds every 2 minutes.
Centrifuge briefly and place on magnet for 1 minute.
Transfer supernatant to a new 2 mL LoBind tube.
Measure DNA concentration with Qubit (2 µL input). Expected yield: 15–40 ng total.
Note
For guidance please refer to:
Josh Quick 2025. DNA Quantification using the Qubit Fluorometer. protocols.io https://dx.doi.org/10.17504/protocols.io.261gekdeyg47/v1

DNA may be stored at 4°C overnight before starting adaptor ligation if needed. 
Adaptor Ligation
Thaw and place on ice block: NEBNext Quick Ligation Buffer, SFB, Quick T4 DNA Ligase, Native Adapter (NA), Elution Buffer (EB).
Vortex Ligation Buffer for up to 39 seconds before use.
In a 2 mL LoBind tube, combine:
30 µL Barcoded pool
5 µL Native adapter (NA)
10 µL NEBNext Quick Ligation Buffer
5 µL Quick T4 DNA Ligase

Incubate at room temperature for 20 minutes.
Add 150 µL AmpureXP beads (3x). Mix by pipetting. Incubate at room temperature for 10 minutes.
Note
Set Thermomixer to 37°C

Centrifuge briefly and place on magnet until liquid is clear.
Remove supernatant and take tube off the magnet.
Add 125 µL SFB and resuspend.
Centrifuge briefly and place on magnet until liquid is clear.
Repeat again:
Remove supernatant and take tube off the magnet.
Add 125 µL SFB and resuspend.
Centrifuge briefly and place on magnet until liquid is clear.
 Remove supernatant. Use a P10 pipette tip to remove the last traces.
Take the tube off the magnet. Add 34 µL EB (black cap) and resuspend.
Incubate tube at 37°C on the Thermomixer for 10 minutes, flicking gently every 2 minutes.
Centrifuge briefly and place on magnet for at least 1 minute.
Transfer supernatant (finished library) to a new 2 mL LoBind tube labeled 'cfDNA library' with today's date on the cap.
Measure DNA with Qubit (2 µL input). Expected yield: ~10 ng total (0.4 ng/µL)
Note
For guidance please refer to:
Josh Quick 2025. DNA Quantification using the Qubit Fluorometer. protocols.io https://dx.doi.org/10.17504/protocols.io.261gekdeyg47/v1

 Store library at 4°C until loading.
Flow cell loading
Allow a PromethION flow cell to reach room temperature for at least 20 minutes.

Thaw, mix, and keep on ice: Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Tether (FCT), Flow Cell Flush (FCF).
Insert flow cell into PromethION 2 Solo. Start flow cell check in MinKNOW.
Note
Check flow cell for condensation and remove any liquid. Verify heat pad is visually intact.

Flow cells must have >5000 pores or must be replaced by Oxford Nanopore. Report within 2 days if below threshold.

Prepare priming mix: add 30 µL FCT to a new FCF tube (1170 µL) for each flow cell to be started. Keep on ice
 Store library at 4°C until loading.Open the priming port (clockwise).
Set a P1000 pipette to 200 µL. Remove air from port by inserting the tip and turning the pipet volumen to 220 µL gently.
Note
Visual flow cell loading guides can be found at the Oxford Nanopore community.
https://www.welcome.nanoporetech.com/

 Load 500 µL priming mix and wait 5 minutes.
Prepare loading mix in a 2 mL LoBind tube:
100 µL SB
68 µL LIB
32 µL Prepared library
Load 500 µL priming mix to complete priming.
 Load 200 µL loading mix with a P1000. Close port and attach light shield.
Wait at least 20 minutes before starting the sequencing
Sequencing setup
The MinKNOW interface has changed multiple times so giving a detailed description of the setup is difficult but start the sequencing run with the following settings added:
Super-accurate Basecalling (SUP)
Do not remove short reads with a filter
PHRED score > 10
Run duration: 96 hours