Jun 04, 2026

Ultra-long DNA Extraction for UMI-Tagged rRNA Operon Amplification Nanopore sequencing V.1

DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjkk8wgk5/v1
  • 1Georgia Institute of Technology
  • Kaiqin Bian
Kaiqin Bian
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjkk8wgk5/v1
Protocol Citation: Kaiqin Bian, Jinhao Yang, Zijun Meng, Ameet Pinto 2026. Ultra-long DNA Extraction for UMI-Tagged rRNA Operon Amplification Nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjkk8wgk5/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 13, 2026
Last Modified: June 04, 2026
Protocol  Integer ID: 316939
Keywords: tagged rrna operon amplification nanopore, rrna operon amplification nanopore, hmw dna recovery, dna extraction, dna extraction for umi, total dna yield, sequencing recovery, dna at sufficient yield, microbial community sample, commercial extraction kit, dna, enzybead, sequential extraction workflow
Funders Acknowledgements:
National Science Foundation
Grant ID: 2428375
Abstract
Recovery of high-molecular-weight (HMW) DNA at sufficient yield is a key prerequisite for long-read amplicon sequencing of the full 16S–ITS–23S rRNA operon, yet most commercial extraction kits are optimized either for yield or for fragment length, rarely both. Here we describe two sequential extraction workflows—DoubleBeads (DB) and EnzyBeads (EB)—that adapt established commercial protocols to simultaneously maximize HMW DNA recovery and total DNA yield from environmental and microbial community samples.

Materials
  1. Benchtop microcentrifuge (capable of 13,000–16,000 × g for 2 mL tube and 4000 x g for 5 mL tube)
  2. Vortex mixer with a horizontal-tube adapter (e.g., Vortex-Genie 2 with adapter 13000-V1-5) for bead beating
  3. Thermomixer or heat block (37 °C, 56 °C, 80 °C)
  4. Magnetic separation rack compatible with 2 mL tubes
  5. Thermal cycler with heated lid
  6. Standard P10/P200/P1000 pipettes and filtered tips
  7. 2 mL and 2.0 mL DNA LoBind microcentrifuge tubes (Eppendorf 022431021 / 022431048)
  8. 0.2 mL PCR tubes or 96-well PCR plates
  9. Qubit Fluorometer (Thermo Fisher) and Qubit assay tubes (Q32856)
  10. 4200 Agilent TapeStation System
  11. HulaMixer‱ Sample Mixer (Thermo Fisher 15920D)
  12. ZymoBIOMICS Gut Microbiome Standard (~3.94 × 10⁹ cells/mL in 2× DNA/RNA Shield) — Zymo Research; Cat. No. D6331
  13. Wizard HMW DNA Extraction Kit (includes HMW Lysis Buffer A, RNase A Solution, Proteinase K Solution, Protein Precipitation Solution) — Promega; Cat. No. A2920
  14. Lysozyme (powder; prepare 10 mg/mL stock in TE) — Thermo Fisher Scientific; Cat. No. 89833
  15. DNeasy PowerWater Kit (includes Solution PW1, Solution IRS, PowerWater Bead Pro Tubes, MB Spin Columns, Solutions PW3, PW4, and EB) — QIAGEN; Cat. No. 14900-100-NF
  16. CleanNGS magnetic beads (SPRI-equivalent) — CleanNA; Cat. No. CNGS-0050 (50 mL)
  17. Platinum SuperFi II Green PCR Master Mix (2×) — used in the UMI-Tagging step (see Note below) — Thermo Fisher Scientific (Invitrogen); Cat. No. 12369010 (50 rxn)
  18. UltraPure DNase/RNase-Free Distilled Water (molecular biology grade) — Thermo Fisher Scientific; Cat. No. 10977015
  19. 1× PBS, pH 7.4, sterile — Thermo Fisher Scientific (Gibco); Cat. No. 10010023
  20. Absolute ethanol, molecular biology grade (for 80% EtOH wash; prepared fresh) — Sigma-Aldrich or equivalent; Cat. No. E7023
  21. Qubit dsDNA HS or BR Assay Kit (for DNA quantification) — Thermo Fisher Scientific (Invitrogen); Cat. No. Q32854 (HS) / Q32850 (BR)
  22. Agilent Genomic ScreenTape kit (for QC: DNA integrity and fragment size measurement) — Agilent; Cat. No. 5067-5366 and 5067-5365


Safety warnings
QIAGEN DNeasy PowerWater Solutions PW1, PW3, PW4, and ethanol are hazardous (flammable and/or contain guanidine salts). Wear lab coat, gloves, and eye protection. Do not mix guanidine-containing solutions with bleach. Refer to all manufacturer SDS documents.
Experimental Design
Sequential DNA extraction methods for ultra-long DNA recovery for UMI-Tagged rRNA operon amplification workflow. ZymoBIOMICS Gut Microbiome Standard is used as a demo.
Method A: EnzyBeads - Promega Wizard HMW (lysozyme + chemical lysis) followed by bead beating (mechanical lysis).
Method B: DoubleBeads - QIAGEN DNeasy PowerWater (mechanical lysis) followed by bead beating (mechanical lysis).

Safety
QIAGEN DNeasy PowerWater Solutions PW1, PW3, PW4, and ethanol are hazardous (flammable and/or contain guanidine salts). Wear lab coat, gloves, and eye protection. Do not mix guanidine-containing solutions with bleach. Refer to all manufacturer SDS documents.
Protocol
Prepare samples
  1. Thaw the ZymoBIOMICS Gut Microbiome Standard (D6331) on ice and invert several times to homogenize.
  2. For each replicate, transfer 150 µL of standard to a 1.5 mL DNA LoBind tube and add 150 µL of 1× PBS. Mix gently by pipetting. This 2× dilution yields a 300 µL working volume.
  3. Optional: Prepare a no-template control (NTC) consisting of 300 µL of 1× PBS only, which iscarried through all downstream steps.
Method A — EnzyBeads (Enzymatic lysis + Bead beating)
  1. Transfer 150 µL of the 2× diluted sample into a 1.5 mL microcentrifuge tube. Centrifuge at 16,000 × g for 2 min to pellet the cells, then remove the supernatant completely (any residual liquid will decrease lysis performance).
  2. Prepare the lysis mixture: 360 µL of 10 mg/mL lysozyme + 360 µL of 10 mg/mL lysostaphin (sufficient for 2 samples in triplicate).
  3. Add 100 µL of lysis mixture and gently pipette to mix. This treatment weakens the cell wall for efficient lysis. Incubate at 37 °C for 60 min.
  4. Add 500 µL of HMW Lysis Buffer A. Using a 1,000 µL wide-bore pipette tip, mix 5 times: draw the contents slowly from the bottom of the tube, then expel the lysate rapidly down the side. The solution should become very viscous. Do not pipette more than 5 times to avoid DNA shearing.
  5. If lysis appears incomplete, incubate at 80 °C for 5 min to complete lysis, then cool to room temperature.
  6. Add 3 µL of RNase A solution to the lysate and mix by inverting the tube 5–7 times. Incubate at 37 °C for 15 min.
  7. Add 20 µL of Proteinase K solution and mix by inverting the tube 10 times. Incubate at 56 °C for 15 min. Cool to room temperature for at least 5 min, or chill on ice for 1 min.
  8. Add 200 µL of Protein Precipitation Solution. Using a 1,000 µL wide-bore pipette tip, mix 5 times (draw from the bottom of the tube, then expel rapidly down the side). Small protein clumps may be visible after mixing. Incubate on ice for 5 min.
  9. Centrifuge at 13,000–16,000 × g for 10 min at room temperature. A protein pellet should be visible. If unpelleted debris remains, repeat the centrifugation.
  10. Slowly transfer the supernatant to a clean 1.5 mL microcentrifuge tube.Note: some supernatant may remain in the original tube with the protein pellet. Leave this residual liquid behind to avoid contaminating the DNA solution with precipitated protein.
  11. Transfer the pellet to a PowerWater Bead Pro Tube and add fresh 1,000 µL of warmed Solution PW1. Vortex at the maximum speed for 2 min.
  12. Centrifuge at 4000 x g for 5 min at room temperature, and collect the supernatant into a clean 2 mL tube.
  13. Combine the supernatant from the first and second lysis steps.
  14. Add 200 μl of Solution IRS and vortex briefly to mix. Incubate at 2–8 C for 5 min.
  15. Centrifuge the tubes at 13,000 x g for 1 min. Avoiding the pellet, transfer the supernatant to a clean 2 ml Collection Tube (provided).
  16. Add 650 μl of Solution PW3 and vortex briefly to mix.
  17. Load 650 μl of supernatant onto an MB Spin Column. Centrifuge at 13,000 x g for 1 min. Discard the flow-through. Repeat until all the supernatant has been processed.
  18. Place the MB Spin Column Filter into a clean 2 ml Collection Tube (provided in the Qiagen PowerWater Kit).
  19. Add 650 μl of Solution PW4 (shake before use). Centrifuge at 13,000 x g for 1 min.
  20. Discard the flow-through and add 650 μl of ethanol (provided) and centrifuge at 13,000 x g for 1 min.
  21. Discard the flow-through and centrifuge again at 13,000 x g for 2 min.
  22. Place the MB Spin Column into a clean 2 ml Collection Tube (provided).
  23. Add 50 μl of Solution EB to the center of the white filter membrane.
  24. Centrifuge at 13,000 x g for 1 min.
  25. Discard the MB Spin Column. The DNA is now ready for downstream applications.

Method B — DoubleBeads (PowerWater-based Bead Beating + Bead Beating)
  1. Warm Solution PW1 (QIAGEN 14900-100-NF) at 55 °C for 5–10 min to dissolve any precipitates; use while still warm.
  2. Transfer 150 µL of the 2× diluted sample into a PowerWater Bead Pro Tube containing 1,000 µL of Solution PW1.
  3. Secure the tube horizontally on a vortex adapter and vortex at maximum speed for 5 min for bead beating.
  4. Centrifuge at ≤ 4,000 × g for 1 min at room temperature.
  5. Transfer the supernatant (~600–650 µL) to a clean 2 mL collection tube.
  6. Centrifuge at 13,000 × g for 1 min to pellet any residual debris.
  7. Avoiding the pellet, transfer the supernatant to a clean 2 mL collection tube.
  8. Add 200 µL of Solution IRS (QIAGEN 14900-100-NF). Vortex briefly to mix. Incubate at 4 °C for 5 min.
  9. Centrifuge at 13,000 × g for 1 min. Transfer the supernatant to a new tube. Retain the bead tube and pellet for the second round of lysis.
  10. Add fresh 1,000 µL of warmed Solution PW1 to the retained PowerWater Bead Pro Tube and vortex at the maximum speed for 2 min.
  11. Centrifuge at 4000 x g for 5 min at room temperature, and collect the supernatant into a clean 2 mL tube.
  12. Combine the supernatant from the first and second lysis steps.
  13. Add 200 μl of Solution IRS and vortex briefly to mix. Incubate at 2–8 C for 5 min.
  14. Centrifuge the tubes at 13,000 x g for 1 min. Avoiding the pellet, transfer the supernatant to a clean 2 ml Collection Tube (provided).
  15. Add 650 μl of Solution PW3 and vortex briefly to mix.
  16. Load 650 μl of supernatant onto an MB Spin Column. Centrifuge at 13,000 x g for 1 min. Discard the flow-through. Repeat until all the supernatant has been processed.
  17. Place the MB Spin Column Filter into a clean 2 ml Collection Tube (provided in the Qiagen PowerWater Kit).
  18. Add 650 μl of Solution PW4 (shake before use). Centrifuge at 13,000 x g for 1 min.
  19. Discard the flow-through and add 650 μl of ethanol (provided) and centrifuge at 13,000 x g for 1 min.
  20. Discard the flow-through and centrifuge again at 13,000 x g for 2 min.
  21. Place the MB Spin Column into a clean 2 ml Collection Tube (provided).
  22. Add 50 μl of Solution EB to the center of the white filter membrane.
  23. Centrifuge at 13,000 x g for 1 min.
  24. Discard the MB Spin Column. The DNA is now ready for downstream applications.
DNA Purification with CleanNGS (Post-Extraction, 0.5× Volume)
  1. Allow CleanNGS beads (CleanNA CNGS-0050) to equilibrate to room temperature for ≥ 30 min and vortex thoroughly until the beads are fully resuspended.
  2. To 50 µL of DNA extracts (input volume), add 25 µL of CleanNGS (0.5× volume ratio). Pipette up and down 15–20 times, or vortex for 30 s.
  3. Incubate on a hula mixer (end-over-end rotation) at room temperature for 5 min.
  4. Briefly spin down. Place the tube on a magnetic rack for ~5 min, until the supernatant is fully cleared.
  5. With the tube still on the magnet, aspirate and discard the cleared supernatant. Do not disturb the bead pellet.
  6. Add 1,000 µL of freshly prepared 80% ethanol. Incubate at room temperature for 1 min. Briefly resuspend the beads by pipetting up and down. Place back on the magnet until cleared, then aspirate and discard the supernatant.
  7. Repeat step 6 for a total of three 80% ethanol wash steps.
  8. Briefly spin down and return to the magnetic rack. Remove all residual ethanol with a fine pipette tip.
  9. Air-dry the bead pellet on the magnetic rack for 5 min. Do not over-dry — cracked pellets reduce recovery.
  10. Remove from the magnet. Add 25 µL of molecular biology grade water or elution buffer. Pipette up and down 20 times, or vortex for 30 s.
  11. Briefly spin down. Incubate at room temperature for 2–3 min.
  12. Place on the magnetic rack until the solution is cleared. Carefully transfer the supernatant (~25 µL) to a clean 200 µL PCR tube.
  13. Quantify the purified DNA with Qubit dsDNA HS Assay (Q32854). .
Quality Control
  1. Quantify the purified DNA extracts with Qubit dsDNA BR Assay (Q32850) following the manufacturer's protocol.
  2. Assess size distribution with TapeStation Genomic DNA ScreenTape following the manufacturer's protocol.
Notes and Troubleshooting
  1. Wide-bore pipette tips are essential for the Wizard HMW kit steps to preserve high-molecular-weight DNA. If unavailable, regular tips can be cut at an angle to widen the bore, or the lysate can be vortexed briefly (5 s). But this will result in some fragmentation.
  2. Always include an NTC (no-template control) carried through every step.
  3. Avoid freeze-thaw of DNA extracts. Store final libraries at 4 °C and use within 1 week, or aliquot before freezing at −20 °C.
Protocol references
[1] Promega Corporation. Wizard® HMW DNA Extraction Kit Technical Manual TM604 (revised 7/2022).
[2] QIAGEN. DNeasy® PowerWater® Kit Handbook (07/2022).
[3] CleanNA. CleanNGS User Manual (current version).
Acknowledgements
The study was funded by the National Science Foundation (NSF) under Award Number 2428375. The authors would also like to acknowledge the School of Civil and Environmental Engineering at the Georgia Institute of Technology for its support of this work.