Dec 11, 2025
ONT Flongle FlowCell Sequening of cfDNA
- 1Hospital de la Santa Creu i Sant Pau
Protocol Citation: Marc Rubio 2025. ONT Flongle FlowCell Sequening of cfDNA. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbwm2qgpk/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: In development
We are still developing and optimizing this protocol
Created: December 11, 2025
Last Modified: December 11, 2025
Protocol Integer ID: 234733
Keywords: ont flongle flowcell sequening of cfdna, using ont flongle flow cell, ont flongle flow cell, custom adaptation for cfdna, ont flongle flowcell sequening, cfdna, microbial cfdna, watchmaker genomics library prep kit, streck tube
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Abstract
This protocol is a custom adaptation for cfDNA sequencing using ONT Flongle Flow Cell, Watchmaker genomics library prep kit and STRECK tubes. It is meant for microbial cfDNA Sequencing.
Protocol materials
Proteinase KThermo Fisher ScientificCatalog #100005393
SDS 20% solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #05030-500ML-F
cfPure Lysis/Binding BufferBioChainCatalog #K5011610-V2
cfPure Magnetic BeadBioChainCatalog #K5011610-V2
cfPure Wash BufferBioChainCatalog #K5011610-V2
cfPure Wash BufferBioChainCatalog #K5011610-V2
cfPure Elution BufferBioChainCatalog #K5011610-V2
ER/AT BufferWatchmaker GenomicsCatalog #7K0101-024
ER/AT Enzyme MixWatchmaker GenomicsCatalog #7K0101-024
Ligation BufferWatchmaker GenomicsCatalog #7K0101-024
Ligation EnzymeWatchmaker GenomicsCatalog #7K0101-024
Ligation Adaptor (LA)Oxford Nanopore Technologies
Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
AMPure XP Beads (AXP) Oxford Nanopore TechnologiesCatalog #SQK-RBK114.96
Short Fragment Buffer (SFB)Oxford Nanopore Technologies
Elution Buffer (EB)Oxford Nanopore Technologies
Flow Cell Flush (FCF)Oxford Nanopore TechnologiesCatalog #SQK-RBK114.96
Flow Cell Tether (FCT)Oxford Nanopore TechnologiesCatalog #SQK-RBK114.96
Flongle Sequencing ExpansionOxford Nanopore TechnologiesCatalog #XP-FSE002
Plasma Isolation from STRECK Tube
30m
Cell-Free DNA BCT is a blood collection tube that stabilizes cell-free
DNA for up to 14 days at 6 °C to 37 °C* and CTCs for up to 7 days at 15
°C to 30 °C*. It is available as a CE marked in vitro diagnostic device
or as a Research Use Only tube, depending on region of use.
300 rpm, 20°C, 00:20:00 , If the tubes were still at room temperature for > 24h you can skip this step. Centrifuge the STRECK tube Sample
Expected result
Expect to see the fases, plasma, plaquetes and leucocytes, and hematites
20m
5 mL Pippete the Plasma into a 15 mL Falcon tube
3000 rpm, 20°C, 00:10:00
Expected result
Debris and Cells in the pellet
10m
Sample Pippete the supernatant into a new Falcon 15 mL tube.
Freeze at-20 °C
Extract cfDNA from plasma isolated from STRECK Tubes using cfPure
46m 10s
Thaw the plasma On ice 1 hour
Add 30 µL Proteinase KThermo Fisher ScientificCatalog #100005393 at 50 mg/mL to each tube
Add 50 µL SDS 20% solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #05030-500ML-F for every 1 mL of plasma.
Incubate at 56 °C for 00:20:00 in the water bath with low shake
20m
After incubation place tubes On ice for 00:05:00 to cool Room temperature
5m
Add 1.25 mL cfPure Lysis/Binding BufferBioChainCatalog #K5011610-V2 for every 1 mL of Plasma
Add 25 µL of cfPure Magnetic BeadBioChainCatalog #K5011610-V2 for every 1 mL of Plasma.
Important: Mix well prior to adding. There should be no visible sedimentation at the bottom of the
solution after mixing.
Adapt the Falcon 15 mL tubes by attaching 2 mL eppendorf using tape
Vortex or 3000 rpm, 00:10:00 on
Equipment
Digital Vortex-Genie 2
NAME
Vortex
TYPE
Scientific Industries, Inc
BRAND
SI-A236
SKU
LINK
at Room temperature
10m
Place tube(s) into a magnet stand 00:03:00 to 00:05:00 or until solution clears.
3m
While keeping the tube(s) on the magnet stand, remove supernatant. Be careful not to
remove magnetic particles
Keep tube(s) on magnet stand for 00:01:00 , and remove residual supernatant
1m
First Wash
Add 1000 µL of cfPure Wash BufferBioChainCatalog #K5011610-V2 to lysis/binding tube(s)
Resuspend beads by vortexing 00:00:10 or pipetting up and down 10 times
10s
Transfer magnetic particle suspension into 1.5 ml micro tube(s) on magnet stand
Allow beads to attach to magnet stand for 10-30 seconds
Pipette supernatant from 1.5 ml tube(s) and use the supernatant to wash the
lysis/binding tube(s)
Transfer the rest of the magnetic particles in lysis/binding tube(s) to the 1.5 ml tube(s)
Keep tube(s) on magnet stand for 10-30 seconds or until solution is clear
Remove as much buffer as possible using a 1000 μl pipette. Tap magnet stand on bench 5 times and remove remaining wash buffer with 200 μl pipette
Transfer tube(s) to non-magnetic rack and add 1000 µL of cfPure Wash BufferBioChainCatalog #K5011610-V2
Resuspend beads by vortexing for 20 seconds or pipetting up and down 10 times
Place tube(s) on magnet stand for 10-30 seconds. Remove as much buffer as possible using a 1000 μl pipette
Second Wash
Transfer tube(s) to non-magnetic rack and add 1000 µL of 80% EtOH
Resuspend beads by vortexing for 20 seconds or pipetting up and down 10 times
Place on magnet stand for 10-30 seconds or until solution clears
Remove as much buffer as possible using a 1000 μl pipette. Remove remaining EtOH with 200 μl pipette
Transfer tube(s) to non-magnetic rack and add 1000 µL of 80% EtOH
Resuspend beads by vortexing for 20 seconds or pipetting up and down 10 times
Place on magnet stand for 10-20 seconds. Remove as much EtOH as possible using a 1000 μl pipette and leave cap open. Remove remaining EtOH with 200 μl pipette. Leave tube(s) open on magnet stand for 00:02:00
2m
Elution Step
Transfer microtube(s) to non-magnetic rack and add 50 µL of cfPure Elution BufferBioChainCatalog #K5011610-V2 and resuspend the beads.
Important: A minimum of 20 μl of cfPure Elution Buffer per ml of plasma is recommended to elute DNA to
ensure optimal yields
Vortex or shake tube(s) vigorously for 00:05:00
5m
Centrifuge tube(s) briefly. And Place tube(s) on magnetic rack for 10 to 30 seconds.
Transfer elute into a new 1.5 ml tube(s
Library Preparation for ONT using WatchMaker
32m
Thaw On ice all reagents and samples
End Repair A-tailing
Prepare End Repair and A-tailing (ER/AT) master mix.
7 µL ER/AT BufferWatchmaker GenomicsCatalog #7K0101-024
3 µL ER/AT Enzyme MixWatchmaker GenomicsCatalog #7K0101-024 for every sample
Mix with pipette up and down
Prepare a 200 µL eppendorf with 25 µL of H2O + 25 µL of cfDNASample
Add 10 µL of ER/AT master mix into 50 µL Sample
Place the tubes in the thermocycler and initiate the end repair and A-tailing program
Proceed immediately to Adapter Ligation after the program has finished and the samples have returned to 4 °C
Adapter Ligation
Prepare the Ligation master mix as follows:
25 µL Ligation BufferWatchmaker GenomicsCatalog #7K0101-024
5 µL Ligation EnzymeWatchmaker GenomicsCatalog #7K0101-024
Add the Ligation master mix 30 µL to the Sample 60 µL and 3 µL ofLigation Adaptor (LA)Oxford Nanopore Technologies from the Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114
Here it says 5 µL but we use 3µL
Place the sample tubes in the thermocycler and initiate the Ligation incubation program
00:15:00
15m
Once the program has completed, proceed immediately to Post-ligation Cleanup
Post-Ligation Cleanup
Thaw SPRI beads AMPure XP Beads (AXP) Oxford Nanopore TechnologiesCatalog #SQK-RBK114.96 On ice to Room temperature
Add 93 µL of AMPure XP Beads (AXP) Oxford Nanopore TechnologiesCatalog #SQK-RBK114.96 to the 93 µL Sample (from thermocycler)
NOTE: SPRI-to-sample bead ratios may be optimized for different applications or adapter configurations. A ratio of 0.8X is recommended as a starting point when using high-quality DNA and full-length adapters. Then 'stubby' adapters are used, the SPRI bead ratio may be increased to 1X (95 μL) to improve performance.
Transfer the mix to a new 1.5 mL eppendorf tube. Incubate the library-bead mixtures at Room temperature for at least 00:05:00 to maximize library recovery.
5m
Place sample tubes on a magnet for at least 00:05:00 , or until all beads have been collected on the tube wall and the solution is clear
5m
Carefully remove and discard the supernatant from each tube
Add 200 µL of Short Fragment Buffer (SFB)Oxford Nanopore Technologies ,taking care to not disturb the bead pellet on the tube wall.
IMPORTANT: Do not use ethanol, use SFB for the cfDNA
Incubate tubes at room temperature for at least 30 sec without removing the tube from the magnet
or disturbing the bead pellet.
Repeat 25.6
Allow remaining Short Fragment Buffer (SFB)Oxford Nanopore Technologies to evaporate by allowing the pellets to air dry for 00:03:00
3m
Remove sample tubes from the magnet and carefully resuspend each bead pellet in 22 µL of Elution Buffer (EB)Oxford Nanopore Technologies . Pipetting carefully will
minimize bubbling and allow for greater library recovery.
Incubate tubes at rRoom temperature for at least 00:02:00 before placing back on the magnet.
2m
Leave sample tubes on the magnet for at least 00:02:00 , or until all beads have collected on the tube wall and the solution is clear
2m
Carefully transfer 22 µL of each library-containing supernatant to a new, labeled tube. If desired, library amplification may be carried out in the same tube in the presence of SPRI beads (See Library Amplification for reaction setup and Appendix A for details regarding PCR in the presence of beads).
Samples can be stored at 4°C for up to 1 week and at -20°C for up to 1 month
ONT Flongle FlowCell Sequencing
Equipment
MinION Mk1C
NAME
Sequencer
TYPE
Oxford Nanopore Technology
BRAND
MIN-101C
SKU
LINK
Equipment
Flongle Flow Cell (R10.4.1)
NAME
Flow cell
TYPE
Oxford Nanopore Technologies
BRAND
FLO-FLG114
SKU
LINK
Prepare Flush Cell Buffer
117 µL Flow Cell Flush (FCF)Oxford Nanopore TechnologiesCatalog #SQK-RBK114.96
3 µL Flow Cell Tether (FCT)Oxford Nanopore TechnologiesCatalog #SQK-RBK114.96
Prepare library for load
Reagent Volume
Sequencing Buffer (SB) 15 µL
Library Beads (LIB) 10 µL
3 - 20 fMol of the DNA library 5 µL
Total 30 µL
from
Flongle Sequencing ExpansionOxford Nanopore TechnologiesCatalog #XP-FSE002
Load the Flongle Flow Cell after FloCell check