Mar 25, 2026
Microbial DNA extraction from seawater filters using the E.Z.N.A.® Water DNA Kit and full-length 16S rRNA gene sequencing on Oxford Nanopore MinION
- Emma Rocke1,
- Nicole Dames2,
- Kathryn Morrissey3
- 1University of Cape Town;
- 2Marine Biological Laboratory;
- 3South African Environmental Observation Network
Protocol Citation: Emma Rocke, Nicole Dames, Kathryn Morrissey 2026. Microbial DNA extraction from seawater filters using the E.Z.N.A.® Water DNA Kit and full-length 16S rRNA gene sequencing on Oxford Nanopore MinION. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4p6p8lo5/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: March 25, 2026
Last Modified: March 25, 2026
Protocol Integer ID: 313889
Keywords: microbial dna extraction from seawater filter, microbial dna extraction, environmental dna suitable for amplicon library preparation, processing of seawater sample, seawater sample, seawater filter, environmental dna, dna extraction, bacterioplankton, rrna gene data for downstream analysis, marine bacterial community composition, rrna gene data, using membrane filtration, membrane filtration, oxford nanopore minion this protocol, oxford nanopore minion, length 16s rrna gene, microbial community analysis, rrna gene, dna yield, seawater, oxford nanopore technologies platform, amplicon library preparation, µm polycarbonate membrane, polycarbonate membrane, filter, organism, extraction
Disclaimer
This protocol is provided as a record of the procedures used in our laboratory and is intended for research use only. It does not represent an endorsement by the kit or instrument manufacturers, nor does it replace the official instructions supplied with any commercial reagents or devices. Users should adapt volumes, incubation times, and instrument settings to their own samples, equipment, and institutional biosafety requirements, and should perform appropriate negative and positive controls to validate performance in their hands. The authors and their institutions accept no liability for any loss, damage, or injury resulting from the use of this protocol; implementation is at the user’s own risk and responsibility.
Abstract
This protocol describes the collection and processing of seawater samples for microbial community analysis using membrane filtration, DNA extraction with the E.Z.N.A. Water DNA Kit, and full‑length 16S rRNA gene sequencing on Oxford Nanopore Technologies platforms. Seawater (5 L per sample) is pre‑filtered through a 200 µm mesh to remove large particles and organisms and then filtered onto 0.2 µm polycarbonate membranes to capture bacterioplankton. Filters are stored frozen prior to extraction, then cut and processed following the manufacturer’s instructions for the E.Z.N.A. Water DNA Kit to obtain high‑quality environmental DNA suitable for amplicon library preparation. DNA yields are quantified with Qubit, with optional clean‑up and concentration for low‑yield samples, and full‑length 16S libraries are generated using the ONT 16S Barcoding Kit (SQK‑16S024) and a high‑fidelity polymerase. Barcoded libraries are bead‑purified, normalized, pooled, and sequenced on MinION flow cells (R9.4.1) to obtain long‑read 16S rRNA gene data for downstream analysis of marine bacterial community composition.
Guidelines
- This protocol uses only the E.Z.N.A.® Water DNA Kit for DNA extraction across all seawater samples to ensure methodological consistency.
- The default PCR enzyme is a high-fidelity master mix (e.g., Q5), and any deviations (e.g., initial use of LongAmp® on a subset of samples) should be clearly annotated in the metadata and manuscript methods.
Materials
**Reagents**
- Seawater (5 L per sample)
- E.Z.N.A.® Water DNA Kit (Omega Bio-tek)
- 200 µm mesh pre-filter
- 0.2 µm polycarbonate membrane filters (suitable for microbiological DNA)
- Nuclease-free water
- Qubit dsDNA BR Assay Kit and/or Qubit dsDNA HS Assay Kit (Thermo Fisher)
- 70% ethanol (molecular biology grade)
**Consumables**
- Sterile carboys or sampling bottles (≥5 L)
- Sterile 1.5 mL or 2.0 mL microcentrifuge tubes
- Sterile scissors or scalpels
- Filter holders and vacuum or peristaltic filtration setup
- Pipette tips (filtered, sterile)
**Equipment**
- Peristaltic pump or vacuum filtration system
- −20°C freezer for filter storage
- Benchtop microcentrifuge
- Vortex mixer
- Heating block or thermomixer (up to 65°C)
- Qubit 4 Fluorometer (or equivalent)
Troubleshooting
Safety warnings
_Note: If any subset of samples was initially amplified with an alternative Taq (e.g., LongAmp®), this should be documented clearly in the 'Notes' section; the majority of samples should be run with the selected high-fidelity polymerase for consistency._
Before start
- Work in a clean area to minimize contamination.
- Pre-label all sample bottles, filters, and tubes.
- Cool seawater samples and process/filtrate as soon as possible after collection (ideally within the same day).
Step 1: Seawater collection
Collect 5 L of seawater per sample into clean, labeled carboys or bottles.
Keep samples shaded and cool (on ice or in a cooler) until filtration. Aim to filter within 24 hours of collection.
Step 2: Pre-filtration
Assemble a 200 µm mesh pre-filter on the filtration apparatus.
Gently pre-filter each 5 L seawater sample through the 200 µm mesh to remove large particles and debris.
Discard the retained material; collect the pre-filtered seawater in a clean reservoir.
Step 3: Filtration onto 0.2 µm polycarbonate filters
Mount a 0.2 µm polycarbonate membrane filter in a sterile filter holder.
Filter the pre-filtered seawater (up to 5 L) through the 0.2 µm filter using vacuum or peristaltic pumping. Adjust flow to avoid filter clogging.
If the filter clogs before the full 5 L is processed, record the final filtered volume in the metadata and, if necessary, use additional filters for the same sample.
When filtration is complete, carefully remove the filter with sterile forceps, fold (cell side inwards), and place it into a labeled sterile microcentrifuge tube.
Immediately freeze filters at −20°C (or preferably −80°C if available) until DNA extraction.
Step 4: DNA extraction using the E.Z.N.A.® Water DNA Kit
Thaw filters on ice briefly just before extraction.
In a clean area, use sterile scissors or a scalpel to cut each filter into small pieces inside a microcentrifuge tube compatible with the E.Z.N.A. Water DNA protocol.
Add the appropriate lysis buffer from the E.Z.N.A. Water DNA Kit (e.g., Buffer MBL or equivalent, depending on kit version) to each tube as per manufacturer’s instructions.
Vortex thoroughly to ensure the filter pieces are fully immersed.
Incubate at the recommended temperature and duration for lysis (typically 65°C for 10–30 min, depending on the kit version).
Proceed through the kit protocol:
- Add binding buffer and ethanol as specified.
- Transfer lysate to the HiBind® DNA spin column.
- Centrifuge and discard flow-through.
- Perform all wash steps with the kit wash buffers.
Dry the column by an additional centrifugation step to remove residual ethanol.
Elute DNA with 50–100 µL of pre-warmed elution buffer (or nuclease-free water) directly onto the membrane, incubate briefly (≈2–5 min), then centrifuge.
Optionally perform a second elution if higher total yield is desired, noting that this will lower DNA concentration.
Store extracted DNA at −20°C until quantification and library preparation.
Step 5: DNA clean-up and concentration (if needed)
Measure DNA concentration with a Qubit 4 Fluorometer using the dsDNA Broad Range (BR) kit.
For low-yield samples, switch to the Qubit dsDNA High Sensitivity (HS) kit to obtain accurate quantification.
If DNA concentration is too low or if inhibitors are suspected (e.g., poor amplification), clean and concentrate the DNA using a column-based DNA clean-up kit (e.g., DNA Clean 6 Concentrator-5, Zymo Research) according to the manufacturer’s protocol.
Elute in a small volume (e.g., 10–20 µL) to increase DNA concentration and re-quantify with Qubit.
Step 6: 16S library preparation (Oxford Nanopore SQK-16S024)
Thaw DNA on ice and dilute or concentrate to approximately 50 ng in 10 µL per reaction.
Prepare full-length 16S rRNA gene libraries using the Oxford Nanopore 16S Barcoding Kit 1–24 (SQK-16S024), following the ONT protocol (version 16S_9086_v1_revH_14Aug2019), with the modification that a high-fidelity Taq master mix (e.g., Q5® High-Fidelity 2X Master Mix) is used as the default polymerase.
PCR reaction setup (25 µL total)
For each sample, assemble on ice:
- 12.5 µL high-fidelity Taq master mix (e.g., Q5® High-Fidelity 2X Master Mix)
- 2.5 µL ONT 16S primer/barcode mix
- 10 µL template DNA (~50 ng)
PCR cycling conditions
Run PCR with the following cycling program:
- 95°C for 1 min (initial denaturation)
- 37 cycles of:
▪ 95°C for 20 s (denaturation)
▪ 55°C for 30 s (annealing)
▪ 72°C for 2 min (extension; high-fidelity Taq)
- Final extension: 72°C for 5 min
Step 7: Amplicon clean-up and pooling
Clean PCR products using a bead-based clean-up protocol (e.g., Mag-Bind® TotalPure NGS or AMPure® XP) according to the manufacturer’s instructions, typically at a 0.8–1.0× bead ratio.
Elute purified amplicons in an appropriate low-EDTA buffer or nuclease-free water (e.g., 10–20 µL).
Quantify cleaned amplicons with Qubit (BR or HS, depending on concentration).
Normalize and pool up to 24 barcoded libraries equimolarly to a final total DNA amount of 50–80 ng for each sequencing run.
Step 8: Nanopore sequencing
Prepare the pooled library for loading on the MinION flow cell (R9.4.1) following the SQK-16S024 protocol (including any required rapid adapter or loading mix steps).
Load the prepared library onto an R9.4.1 flow cell on a MinION Mk1B or Mk1C device.
Run each sequencing experiment for approximately 16 hours, monitoring pore health and yield via MinKNOW.
After each run, proceed with basecalling and demultiplexing as per ONT recommendations for 16S barcoded libraries.