Feb 19, 2025

Public workspaceExtraction and Sequencing of High Molecular Weight Genomic DNA from Auxenochlorella protothecoides using the Oxford Nanopore Technologies MinION Platform.

DOI
dx.doi.org/10.17504/protocols.io.14egn6r8ql5d/v1
  • 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA;
  • 2Quantitative Biosciences Institute, University of California, Berkeley, California 94720, USA
  • Merchant Lab UC Berkeley
Dimitrios J. Camacho
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DOI: dx.doi.org/10.17504/protocols.io.14egn6r8ql5d/v1
Protocol Citation: Dimitrios J. Camacho, Rory J. Craig, Sabeeha S. Merchant 2025. Extraction and Sequencing of High Molecular Weight Genomic DNA from Auxenochlorella protothecoides using the Oxford Nanopore Technologies MinION Platform.. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6r8ql5d/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: August 20, 2024
Last Modified: February 19, 2025
Protocol Integer ID: 106051
Keywords: Oxford nanopore, nanopore, minION, Mk1B, genomic DNA, high molecular weight DNA, UTEX 250, Auxenochlorella, MinKnow, Dorado, algae, green algae, DNA extraction, sequencing, whole genome sequencing
Funders Acknowledgements:
National Institutes of Health (NIH): Molecular Basis of Cell Function T32 Training Grant
Grant ID: 5T32GM007232-44
US Department of Energy (DOE), Office of Biological and Environmental Research (BER): Systems Engineering of Auxenochlorella protothecoides: from Photosynthesis to Biofuels and Bioproducts Grant
Grant ID: DE-SC0023027
University of California, Berkeley, Chancellor’s Fellowship
Grant ID: N/A
US Department of Energy (DOE), Lawrence Berkeley National Laboratory, Laboratory Directed Research and Development Program
Grant ID: DE-AC02-05CH11231
Disclaimer
The "High molecular weight DNA extraction" section of this protocol was adapted from: Frédéric Chaux-Jukic, Nicolas Agier, Stephan Eberhard, Zhou Xu 2024. Extraction and selection of high-molecular-weight DNA for long-read sequencing from Chlamydomonas reinhardtii. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59j9jg1b/v2
Document version: Version 2, updated February 14, 2024

The "DNA size selection using the Short Read Eliminator (SRE) kit" section was adapted from PacBio's Short Read Eliminator (SRE) kit manual (v02), which can be found here: https://www.pacb.com/wp-content/uploads/Procedure-checklist-Removing-short-DNA-fragments-with-the-Short-Read-Eliminator-SRE-kit.pdf
Document version: Version 02, released March 2024

The "End-prep and nick repair", "Ligation of sequencing adapters", "Priming the flow cell", and "Loading the library into the flow cell", sections of this protocol were adapted from Oxford Nanopore's "Ligation Sequencing DNA V14 (SQK-LSK114)" manual, which can be found here: https://nanoporetech.com/document/genomic-dna-by-ligation-sqk-lsk114?device=MinION
Document version: GDE_9161_v114_revW_29Jun2022

The "Washing and reloading the flow cell" section of this protocol was adapted from the "Recover a library to replace on a washed MinION flow cell" section of Oxford Nanopore Technologies, "Library recovery from flow cells" manual, which can be found here: https://nanoporetech.com/document/library-recovery-from-flow-cells#recover-a-library-to-replace-on-a-washed-minion-flow-cell
Document version: LIR_9178_v1_revJ_11Jan2023

The manuals listed above are updated frequently. When ordering kits, please refer to the associated manuals with the latest updates.
Abstract
This protocol describes a method for extracting and sequencing high molecular weight DNA from Auxenochlorella protothecoides (UTEX 250). The protocol incorporates DNA purification steps where RNA is degraded, larger DNA fragments (>10 kb) are enriched, and DNA nicks are repaired. Sequencing libraries are prepared from the resulting high molecular weight DNA using the Ligation Sequencing Kit (version 14 chemistry) and sequenced using the Oxford Nanopore Technologies MinION (Mk1B model). POD5 files are generated, and bases are called using the MinKnow’s built-in Dorado base caller. With just a single flow cell, the entire genome of Auxenochlorella protothecoides (approximately 44 Mbp in diploid size) can be sequenced at a coverage depth of 50x. This method reliably produces 30 kb (N50) reads which can be used to resolve repetitive regions, assess large structural rearrangements, and detect off target integrations of gene editing cassettes. Additionally, methylated bases such as 5mC, 5hmC, and 6mA can be distinguished from canonical bases using the Dorado base-caller, thus allowing for the analysis of epigenetic methylation profiles.
Guidelines
High molecular weight genomic DNA is fragile and susceptible to shearing from mechanical stress. For this reason, pipetting and bead beating are not recommended. If pipetting is required, cut pipette tips off with scissors to generate a larger orifice or purchase nuclease-free wide-orifice tips. Avoid vortexing DNA and enzymes. Flick tubes gently to mix instead.
 
Do not freeze high molecular weight DNA. Store DNA at Temperature4 °C . If storing DNA for extended periods of time, flash freeze DNA in liquid nitrogen and freeze at Temperature80 °C . 
 
Check the compatibility of components. This protocol utilizes components compatible with Oxford Nanopore Technologies' (ONT) version 14 chemistry only.
Materials
Equipment:
RAININ P1000 pipette and low adhesion tips
RAININ P200 pipette and low adhesion tips
RAININ P20 pipette and low adhesion tips
RAININ P2 pipette and low adhesion tips
Timer
Ceramic mortar and pestle
Flexible plastic spatulas
Centrifuge, model JXN-26, Beckman Coulter Avanti (with JA14.50 fixed angle rotor)
Centrifuge, model 5810 R, Eppendorf, (with A-4-81 15/50 mL swinging bucket rotor) 
Centrifuge, model 5424R, Eppendorf, (with FA-45-24-11 2 mL fixed angle rotor)
Vortex mixer, Corning, REF 6775
Magnetic separator rack for 1.5 mL Eppendorf tubes
Qubit fluorometer, Thermofisher Scientific, model: 
Nanodrop, Thermofisher Scientific, model: NanoDrop One / NanoDrop One C
MinIon, Oxford Nanopore Technologies, model: Mk1B
Femto Pulse system
Temperature50 °C water bath or heating block, PolyScience WBE02
Temperature65 °C water bath or heating block, PolyScience WBE02
Temperature37 °C water bath or heating block, PolyScience WBE02
Sterile laminar flow hood
Fume hood

Computer hardware and software requirements 
Software: MinKnow 24.06.5 or later
Operating systems: Windows 10 or later, MacOS Monterey (12) or later, Linux Ubuntu 20.04 or later.
RAM: 16 GB or more.
USB port: A USB 3.0 port, USB Type-A or USB Type-C (5.0 Gb/s, or faster transfer speed)
CPU: Intel i7, i9, Xeon, or better with at least 4 cores/ 8 threads, Apple Silicon M1, M2, or better.
GPU: NVIDIA GPU RTX 4070 or better, Apple silicon M1, M2, or better.
Storage: 1 TB of free storage or more on a Solid State Drive (SSD). 
 
Kits:
dsDNA Broad range kit, ThermoFisher Scientific (Cat. No. Q32850)
Ligation Sequencing Kit V14, Oxford Nanopore Technologies (Cat No. SQK-LSK114)
Short read eliminator (SRE) kit (Size selection of DNA > 25 kb), PacBio (Cat No. 102-208-300)
NEBNext‱ Companion Module v2 for Oxford Nanopore Technologies Ligation Sequencing NEB# E7672 (Cat. No. E7672S)

Note on kits: Please ensure that flow cells and all kits are compatible with Oxford Nanopore's version 14 chemistry.
 
Reagents and chemicals:
Hexadecyltrimethylammonium bromide, molecular biology reagent (CTAB), MP Biomedical (Cat. No. 194004)
Sodium Chloride, Fisher Scientific, (Cat. No. BP358-212)
Ethylenediamine Tetraacetic Acid, Disodium Salt Dihydrate, (EDTA), Fisher Scientific, (Cat. No. S311-500)
Polyvinylpyrrolidone (PVP) (Average molecular weight = 40,000 g/mol) Sigma-Aldrich, (Cat. No. PVP40-50G)
Tris Base, Fisher Scientific, (Cat. No. BP152-1)
Hydrochloric acid, Fisher Scientific, (Cat. No. A144S-212)
Monarch DNase and protease free RNase A, [20 mg/ mL], 1 mL, New England Biolabs (Cat. No. T3018L)
Proteinase K Solution, Molecular Biology Grade, 20 mg/mL MP Biomedical (SKU: 0218398810)
Phenol: chloroform: isoamyl alcohol (25:24:1), 1 M Tris, Fisher Scientific, (Cat. No. BP1752I-400)
Ethylenediamine Tetraacetic Acid, Disodium Salt Dihydrate, (EDTA), Fisher Scientific, (Cat. No. S311-500)
100% ethanol, Sigma-Aldrich, (Cat. No. E7023-1L)
Fresh 70% ethanol
Milli-Q H2O
AMPure XP beads, Beckman Coulter (Cat. No. A63880)

Consumables:
MaXtract High Density Tubes, 100 x 15 mL, QIAGEN, max rating: 3,500 ×g (Cat. No. 129065)
Note
The MaXtract High Density Tubes have been discontinued. You can create your own phase-lock tubes using regular 15 mL tubes and Dow High Vacuum Grease. (Mukhopadhyay & Roth, 1992) See https://bitesizebio.com/18944/diy-phase-separating-gel-clean-and-cheap/

Diamond Max Centrifuge Tube, 50ml, 25/Re-Sealable Bag, Polypropylene,(Max rating of 20,000 ×g), Globe Scientific, (Cat. No. 6297)
1.5 mL Eppendorf DNA LoBind microfuge tubes, FIsher Scientific (Cat. No. 13-698-791)
Flow Cell (R10.4.1) Oxford Nanopore Technologies, (Cat.No. FLO-MIN114)
Safety warnings
This protocol uses phenol and chloroform, which are toxic if ingested, inhaled, or absorbed through skin.
Use a lab coat, eye protection, closed toe shoes, pants, and gloves when working with phenol and chloroform. Perform all work with phenol and chloroform in a fume hood. Receive the proper training before handling these chemicals. Ensure that all phenol and chloroform hazardous waste is properly labeled, stored, and disposed of.

Liquid nitrogen is a cryogenic that may cause severe burns. Use proper PPE including cryogenic gloves, closed toe shoes, pants an apron, a face shield, and a lab coat. Liquid nitrogen may displace oxygen and cause asphyxiation. Do not accompany liquid nitrogen dewars over 10 liters in an elevator.
Before start
  1. Make Amount1 L of 1 M Tris-HCl, pH 8.
a. Add Amount800 mL of nuclease free water to a 1 L bottle.
b. Add Amount121.14 g of Tris Base and mix.
c. Adjust to pH 8 by adding HCl.
d. Fill to Amount1 L with nuclease free water.

2. Make Amount1 L of 0.5 M Na2EDTA-NaOH, pH 8.
a. Add Amount800 mL of nuclease free water to a 1 L bottle.
b. Add Amount186.12 g of Na2EDTA and mix.
c. Adjust the pH to 8 by adding NaOH. EDTA will not dissolve unless the pH is adjusted to pH 8.
d. Fill to Amount1 L with nuclease free water.

3. Make CTAB Buffer
a. Fill a bottle with Amount600 mL of nuclease free water.
b. Add Amount20 g of CTAB and mix.
c. Add Amount10 g of PVP and mix.
d. Add Amount50 mL of 1 M Tris-HCl, pH 8. Mix well.
e. Add Amount81.8 g of NaCl and mix.
f. Add Amount40 mL of 0.5 M Na2EDTA-NaOH, pH 8.
f. Fill to Amount1 L with nuclease free water and mix well.
f. Heat CTAB at Temperature65 °C to solubilize precipitates right before use.
 
4. Make wide orifice pipette tips by cutting the dispensing end of pipette tips.

5. Prepare wet ice in an ice bucket. 

6. Cool the Eppendorf 5424R microfuge to Temperature4 °C .

7. Heat Temperature65 °C water bath.

8. Heat Temperature37 °C water bath.

9. Make Amount50 mL of fresh 70% ethanol. See note below.

10. Make RNase A aliquots. See below.
a. DNase free RNase is supplied as [20 mg/ mL] in 1 mL. Each sample is treated with Amount25 µL of RNase A twice (steps 8 and 11).
b. Make Amount51 µL aliquots and store at Temperature-20 °C . Thaw one aliquot/ sample on wet ice right before use.


11. Make Amount11 µL aliquots of the proteinase K solution. Thaw one aliquot/ sample on wet ice right before use.

12. Fill a Amount5 L dewar with liquid nitrogen.

13. Thaw AMPure XP beads at room temperature. Make aliquots if necessary to avoid freeze-thaw cycles.
It is very important that the beads remain in solution. Store the containers and tubes upright and spin them down quickly before storage. The beads will crack if dry, resulting in a loss of DNA binding affinity and introduction of impurities.

14. Ensure that your computer is capable of data acquisition (see materials for hardware and software requirements).

15. Prepare the proper hazardous waste containers for phenol and chloroform.

16. Make fresh 80% ethanol. See note below.

17. Set a thermal cycler program for Temperature20 °C for 5 min thenTemperature65 °C for 5 min. Amount60 µL reaction volume.

18. Perform a flow cell check to ensure that your flow cells have at least 800 active pores. Flow cells have an expiration date and warranty period.

19. Prepare 2 QIAGEN MaXtract tubes/ sample. 
a. Centrifuge new MaXtract tubes at 3,000 ×g for 2 min before use. Ensure that the MaXtract High Density gel has settled on the bottom of the tube without any air bubbles. If not, centrifuge again.

Note: Always prepare fresh diluted ethanol from 100% ethanol. Ethanol has a lower boiling point and will evaporate before water, which may result in a lower percentage of ethanol. Diluted ethanol will not precipitate DNA as efficiently and will lead to loss of DNA yield.

High molecular weight DNA extraction
High molecular weight DNA extraction
Grow Auxenochlorella protothecoides cells in a liquid medium to mid-log growth phase (107 cells / mL).
Determine the cell density and volume required to collect more than 108 cells. (Camacho & Merchant, 2024)
Transfer up to Amount45 mL of liquid culture to a 50 mL Diamond Max Centrifuge Tube. These tubes are designed to withstand high centrifugal forces (20,000 ×g).
Place the tubes into the fixed angle JA-14.50 rotor of the Beckman Coulter Avanti JXN-26 centrifuge.
Centrifuge at 10,000 ×g for 2 min, room temperature.
Discard the supernatant.
Resuspend the cell pellet in Amount500 µL of nuclease free water by gentle pipette mixing (using a wide orifice or cut pipette tip).
Use a spoil board to insulate the lab bench from liquid nitrogen. Cryogenic liquids may cause lab benches and sinks to crack.

Cool the mortar. Put a ceramic pestle in a ceramic mortar and fill the mortar with liquid nitrogen. Wait for the liquid nitrogen to boil off and fill the mortar again.

Repeat (3–5×) until the mortar and pestle are chilled and liquid nitrogen does not boil off violently.

Fill the mortar once more.



Using a P1000 with a wide orifice pipette tip, slowly add the cell suspension to the liquid nitrogen in a drop wise manner. Smaller drops are better. The drops will freeze in the liquid nitrogen. Once all the suspension is in the mortar, wait for the liquid nitrogen to boil off.
Note
Although wide orifice tips are not required at this step, they make it easier to pipette the viscous cell suspension. You may also find it easier to start crushing while the drops of culture are still suspended in liquid nitrogen. This decreases the chance of fragments flying out of the mortar but increases the chances of liquid nitrogen splashing. Please be cautious and wear the proper PPE when working with liquid nitrogen.

If you allow the mortar and pestle to warm up, the cell suspension will melt, and crushing will be much more difficult due to the cell suspension sticking to the mortar and pestle.



Begin pulverizing the frozen cell suspensions with light tapping. Fragments of the frozen cell suspension may fly out of the mortar, so tap lightly. Re-apply liquid nitrogen to the mortar and pestle if necessary. Once all large frozen fragments are broken, start grinding slowly in a circular motion. The final product should look like green matcha powder if the cultures were green.



Scrape the powder into a 50 mL falcon tube with a plastic spatula.
Add Amount2 mL of preheated CTAB buffer to the powder.
Add Amount5 µL of proteinase K (provided at 20 mg ⋅ mL−1) and Amount25 µL of DNase-free RNAse A (provided at 20 mg ⋅ mL−1). Mix by gentle inversion and incubate at Temperature65 °C 
for 30 min.

Safety information
Do not inhale phenol and/or chloroform.
Perform all work with phenol and chloroform in a fume hood (parts of steps 10–16).
Tubes containing phenol and/or chloroform should only be opened in the fume hood.
Decant the solution to a 15 mL phase-lock gel tube (QIAGEN MaXtract).


Use a serological pipette to carefully pierce through the top layer of TRIS and draw up the phenol:chloroform:isoamyl alcohol from the bottom the layer.

Add Amount2 mL of room temperature phenol:chloroform:isoamyl alcohol (25:24:1). Mix by gentle inversion (~40 turns per minute) for 5 min.
Ensure that the MaXtract High Density tubes are prepared (see the "Before Start" section)
Centrifuge at 1,500 ×g for 5 min at room temperature using the A-4-81 swinging bucket rotor of the Eppendorf 5810 R centrifuge.

Slowly lift a tube out of the rotor and check if the phases are clearly separated. If not, centrifuge for an additional 2 min. If the phases are clearly separated, place the tubes on a rack and set the centrifuge to Temperature4 °C . Close the lid to allow the centrifuge to cool.



Decant the top layer (aqueous phase containing nucleic acids) to a new 15 mL phase-lock gel tube.

The middle layer should contain a white gel, which was used to separate the top aqueous layer from the bottom organic layer (containing phenol and chloroform).

Set the MaXtract phase-lock gel tubes aside in a phenol/ chloroform waste container. You may need to separate phenol and chloroform liquid waste from contaminated solid waste later. See step 36.
Add Amount5 µL of proteinase K and Amount25 µL of RNAse A to the aqueous phase in a new MaXtract phase-lock tube. Mix gently by inversion and incubate at Temperature37 °C for 20 min.
Add Amount2 mL of room temperature phenol:chloroform:isoamyl alcohol (25:24:1). Mix by gentle inversion (~40 turns per minute) for 5 min.
Centrifuge at 1,500 ×g for 5 min at Temperature4 °C using the same rotor and centrifuge as step 11.
Add 2 mL of chloroform:isoamyl alcohol (24:1). Gently mix by inversion for 5 min and centrifuge at 1,500 ×g for 5 min at Temperature4 °C using the same rotor and centrifuge as steps 11 and 14.
Decant aqueous phase to a new 15 mL Falcon tube. Add 2× volume (Amount4 mL ) ice-cold 100% ethanol, slowly mix by inverting 10 times, and incubate on ice for 1 h.
Carefully decant the mixture (including any precipitates formed) into four 1.5 mL DNA lo-bind tubes and centrifuge at 20,000 x g for 10 min at Temperature4 °C using the FA-45-24-11 2 mL fixed angle rotor in a microfuge, Eppendorf model 5424R.
Discard supernatant in each tube and wash with Amount1 mL freshly prepared 70% ethanol (using ultrapure water). Invert gently and centrifuge at 20,000 ×g for 1 min using the same centrifuge and rotor as in step 17. Discard the supernatant and repeat.
After decanting the majority of the supernatant following the second wash, pulse-centrifuge the tubes (use the FA-45-24-11 2 mL fixed angle rotor and hold the "short" button on the 5424R microfuge for 10 s). Pipette off the remaining ethanol.
Dry the pellet in a laminar flow hood for ~5 min.
Resuspend pellets overnight in Amount25 µL of ultrapure water.
Combine four tubes by gently pipetting from three of the tubes into one tube (ideally the one that contained most of the DNA precipitate).
Add an equal volume (Amount100 µL ) of AMPure XP beads, mix gently by hand for 5 min.
 Let the beads reach room temperature beforehand, and vortex for 10 s prior to use.
Place the tube on a magnetic rack for 5 min. Wait for the supernatant to become clear. Discard the supernatant.
Magnetic separator rack for 1.5 mL Eppendorf tubes. Tubes may need to be lifted slightly to allow the magnetic rack to attract the beads to the bottom of the tubes.

Add Amount500 µL 70% EtOH to the tube on the magnetic rack, wait 1 min and remove supernatant.
Remove the tube from the magnetic rack. Pulse-centrifuge the pellet (see step 19).
Pipette off residual ethanol and dry. Do not over-dry the pellet. 30 s–1 min is sufficient.
Residual < ~ Amount5 µL of ethanol is ok.
Resuspend the beads in Amount60 µL of nuclease free water for at least 30 min at room temperature.
Place the tube on the magnetic rack for 5 min or until the supernatant is clear.
Transfer the supernatant containing DNA to a new 1.5 mL lo-bind tube.
Measure the DNA concentration by broad range dsDNA Qubit.
Adjust to ~ 150 ng/µL in Amount60 µL with nuclease free water and proceed to the DNA size selection section.
DNA size selection using the Short Read Eliminator (SRE) kit
DNA size selection using the Short Read Eliminator (SRE) kit
Bring all SRE kit solutions to room temperature. Set the centrifuge to Temperature29 °C .
Add Amount60 µL of SRE buffer to the sample and invert to mix several times.
Incubate the tube at Temperature50 °C for 1 h in a water bath or heating block.
Load the tubes into the centrifuge with the hinge of the tube facing outward and centrifuge at 10,000 ×g for 30 min at Temperature29 °C .
Carefully remove the supernatant with a P200. DNA pellet may not be visible. It is OK to leave up toAmount10 µL of liquid.
Add Amount50 µL of Buffer LTE and incubate at room temperature for 20 min.
Flick the tube to fully resuspend pellet in the buffer.

Optional: Store DNA in Temperature4 °C overnight.
Perform the final QC using a Nanodrop to obtain the A260/A230 and A260/A280 ratios, Qubit to accurately determine the DNA concentration, and gel electrophoresis or femto-pulse capillary electrophoresis to determine the DNA fragment size distribution.

The expected yield is ~6 µg (60% – 85% of starting DNA).
Clean up by consolidating all phenol and chloroform waste.
Some institutions may require liquid and solid phenol chloroform waste to be separated.
The phase-lock tubes will have phenol and chloroform waste locked in the lower phase.
Face the opening of the tube away from you and into the liquid waste container.
Use a P1000 to very carefully pierce the gel.
Pressure exerted by piercing the gel phase may cause the phenol and chloroform to splash out.
Dump the liquid waste into a properly labeled waste container and place the contaminated lab debris in a properly labeled and double bagged waste bag.
End-prep and nick repair
End-prep and nick repair
Thaw or chill all reagents on wet ice:
Nuclease free water
DNA
NEBNext FFPE DNA Repair Buffer v2
NEBNext FFPE DNA Repair Mix
Ultra II End-prep Enzyme Mix
Freshly prepared 80% ethanol

Thaw an aliquot of AMPure XP Beads at room temperature.
Flick all tubes to mix. Do not vortex tubes.
Transfer Amount1 µg of DNA to a 0.2 mL PCR tube.
Adjust the volume to Amount47 µL with chilled nuclease free water and add the following components in the spedified order. Mix after adding each component by gentle flicking, followed by a quick spin.
Add Amount7 µL of NEBNext FFPE DNA repair buffer v2.
Add Amount2 µL of NEBNext FFPE DNA repair mix.
AddAmount3 µL of Ultra II End-prep Enzyme mix.

Use a thermal cycler to incubate the reaction at Temperature20 °C for 5 min and then Temperature65 °C for 5 min.

Transfer the end prep reaction to a clean 1.5 mL LoBind tube.
Resuspend the AMPure XP beads by vortexing. The beads will settle quickly, separating from the mixture. Always resuspend the beads in solution right before drawing the mixture.
Add Amount60 µL of beads to the end prep reaction.
Mix by flicking the tube.

Mix the sample by gentle inversion for 5 min (~40 turns per minute).
Spin the sample down and place the tube on a magnetic rack until the supernatant is clear.
Keep the tube on the magnet and carefully pipette off the supernatant.
Slowly add Amount200 µL of freshly prepared 80% ethanol to the pellet.
Do not disturb the pellet.

Carefully remove the supernatant.
Spin the tube down and place it back on the magnetic rack.
Remove the residual ethanol.
Do not let the pellet dry for over 1 min. The beads may crack.
We find that one wash is sufficient.

Remove the tube from the magnetic rack and add Amount61 µL of room temperature nuclease free water.
Incubate the sample in a Temperature37 °C heat block or water bath for 5 min.
Place the tube back on the magnetic rack and wait until the beads form a compact pellet and the supernatant is clear.
Transfer the supernatant to a clean 1.5 mL LoBind tube.
Quantify the DNA using a Qubit fluorometer.
The recovery may be about 50% of the starting concentration measured by Qubit in step 35.
You may choose to immediately proceed to the next step, or store the DNA at Temperature4 °C overnight.

Ligation of sequencing adapters
Ligation of sequencing adapters
Spin down the Ligation Adapter (LA) and Salt-T4 DNA Ligase. Place on wet ice.
Thaw the Ligation Buffer, Elution Buffer, and Long Fragment Buffer at room temperature. Use the Ligation Buffer (LNB) that is supplied by Oxford Nanopore Technologies.
Pipette mix the Ligation Buffer. Vortex the Elution Buffer and Long Fragment Buffer.
Place all reagents on wet ice.
Add Amount5 µL of Ligation Adapter to the Amount60 µL of DNA eluted from step 48. Mix by gentle flicking.
Add Amount25 µL of Ligation Buffer. Mix by gentle flicking.
Add Amount10 µL of Salt-T4 DNA Ligase. Mix by gentle flicking.

Incubate the reaction at room temperature for 10 min.
Add Amount40 µL of resuspended AMPure XP Beads to the reaction.
Mix by gentle inversion for 5 min at room temperature. Invert 40 times / min.
Spin the sample down and place the tube on the magnetic rack.
Wait for the solution to become clear and pipette off the supernatant.
Add Amount250 µL of Long Fragment Buffer.
Flick the tube to mix.
Spin the tube down.
Place the tube back on the magnetic rack and wait for the supernatant to become clear.
Carefully remove the supernatant. Use a P20 to remove as much ethanol as possible.
Dry the pellet for less than 30 s. Residual <Amount5 µL of ethanol is ok. Do not over dry the pellet.
Remove the tube from the magnetic rack and add Amount15 µL of Elution Buffer.
Incubate the sample at Temperature37 °C for 10 min.
Place the tube back on the magnetic rack and wait until the supernatant is colorless.
Transfer the supernatant to a new 1.5 mL LoBind tube.
Quantify DNA using the Qubit fluorometer.
Use gel electrophoresis or femto-pulse capillary electrophoresis to determine the DNA fragment size distribution.
Dilute the library to 300 ng in Amount12 µL (25 ng/µL) using the Elution Buffer.
Priming the flow cell
Priming the flow cell
Thaw the following reagents at room temperature:
Sequencing Buffer (SB)
Library Beads (LIB)
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)
Bovine Serum Albumin (BSA)
Vortex and spin down all tubes.
Add Amount1170 µL of Flow Cell Flush to a new nuclease free 1.5 mL tube.
Add Amount5 µL of Bovine Serum Albumin. Mix by pipetting.
Add Amount30 µL of Flow Cell Tether Mix by pipetting.
Connect the Mk1B sequencing device to a computer.
Please note that a power button does not exist and the device will power on when connected.

Laptop computer and Mk1B sequencing device. See the materials section for computer hardware requirements.

Obtain a flow cell that has recently passed a flow cell check.
Open the Mk1B device lid and slide the MinION flow cell under the clip. Two guide pins under the flow cell should slide into the pin holes on the Mk1B device.
Ensure correct thermal and electrical contact by pressing down on the flow cell lightly.






Open the flow cell priming port by sliding the cover clockwise.



Check for an air bubble in the priming port after the cover has been moved.
To remove air bubbles, set a P1000 pipette to Amount200 µL and attach a clean empty pipette tip.
Insert the tip into the priming port and carefully turn the dial on the pipette toAmount220 µL to Amount230 µL to draw up Amount20 µL to Amount30 µL . Do not draw more than Amount30 µL .


Note
Irreversible damage to the pores can occur if they are exposed to air.
Visually inspect the priming port and spotON sample port for air bubbles each time the covers are opened.


Load Amount800 µL of the priming solution from step 61 into the priming port.
Avoid introducing air bubbles by keeping a small amount of liquid in the pipette tip.
Do not depress the plunger past the second stop and blow out into the priming port.
Wait 5 min.
Lift the SpotON sample port cover.
Load Amount200 µL of the priming mix into the flow cell priming port, not the SpotON sample port.

Loading the library into the flow cell
Loading the library into the flow cell
Add Amount37.5 µL of Sequencing Buffer to a new 1.5 mL LoBind tube.
Library Beads will settle. Resuspend the beads by pipette mixing.
Add Amount25.5 µL of Library Beads to the Sequencing Buffer in the 1.5 mL tube.
Add Amount12 µL of DNA from step 59 to the mixture.
Mix the library by gently pipetting up and down.
Slowly add Amount75 µL of the library to the SpotON sample port in a dropwise manner.
Watch each drop flow into the port before adding another drop.
Replace the SpotOn sample port cover.
Close the priming port.
Place the light shield above the sensor array.
Close the MinION lid.
MinKNOW software operation
MinKNOW software operation
Open the MinKNOW software on your computer. The software will fail to launch if the computer is not connected to the internet. Telemetry data is shared with ONT and offline MinKNOW operation should be requested from ONT in advance.
MinKNOW Home Screen. Click on "Start" icon to the left and click on "Start sequencing" highlighted in green.

Input experimental details. The flow cell ID can be found on the flow cell itself and is sometimes automatically recognized by the software.
Step 1. Positions

Select the library preparation kit used: Ligation Sequencing Kit SQK-LSK114
Step 2. Kits

Set-up the run configuration.

Note
We recommend enabling Fast basecalling during sequencing if your computer has sufficient GPU; otherwise, you may disable basecalling entirely. In each case, a more accurate and computationally intensive basecalling process will be required after the run. To ensure compatibility with advanced basecalling algortihms, it is essential to select POD5 as the output file format, rather than FAST5 or FASTQ. POD5 files store raw ionic current signal data, offering greater flexibility for interpretation as base callers continue to evolve.


Turn off alignment and adaptive sampling.
Choose when to stop the run.
Click on the green "Start" icon on the bottom right of the screen to start the sequencing run.
Step 3: Run configuration

Click on the "Sequencing overview" tab on the left.
The device will heat the flow cell to 35°C before a pore scan is conducted.
Click on the white flow cell icon below.

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A quick overview of the experiment is shown in each row. Only one experiment is shown below.
Next, click on the experiment.

A detailed description of the experiment will appear in a window below.
Click on the arrows to the left and right to toggle between 13 different instrument panels.
Use the circles on the bottom of the window to quickly navigate to different panels.

Panels available:
Sequencing overview
Channel states
Pore activity
Pore scan results
Cumulative output
Read length histogram
Translocation speed
Q score histogram
Q score over time
Temperature history
Bias voltage history
Disk write performance
Traceviewer
To end the run, click on the stop icon in the run controls section on the top of the screen.
Select which run to stop.
If you plan to wash the flow cell (next section) do not stop the run, instead pause the run.


Washing and reloading the flow cell
Washing and reloading the flow cell
The amount of active pores will decrease throughout the run, partially due to blockages of the pores.
It is possible to recover the original library, wash the flow cell, and reload the original library onto the washed flow cell to increase the coverage of the genome.
Oxford Nanopore Technologies recommends that a library is only transferred 2–3 times max.
A single R10.4.1 flow cell can be washed up to five times and can be used to sequence a different library after each wash.
Place a tube of Wash Mix on ice. Do not vortex the tube.
Thaw a tube of Wash Diluent at room temperature. Vortex to mix. Place on ice.
Pause the sequencing run on the MinKNOW software.
Open the lid of the Mk1B device and slide open the priming port cover (clockwise).
Refer to step 64 to ensure that no air bubbles are present under the priming port cover and sensor array.



Depress the plunger on your pipette before inserting the tip into the priming port.
Slowly and carefully pipette Amount150 µL of the DNA library from the priming port.
Transfer the recovered DNA to a new 1.5 mL LoBind tube and keep it on wet ice.
In a new 1.5 mL LoBind tube, add Amount2 µL of Wash Mix and Amount398 µL of Wash Diluent.
Mix well. Do not vortex the tube.
Close both the priming port cover and the SpotON sample port cover to ensure that air does not enter the sensor array.
Use a P1000 to remove all fluid from the Waste Port 1.
Ensure that no fluid leaves the sensor array.



Open the priming port cover.
Check for and remove air bubbles in the priming port. See step 64.
Add ~Amount400 µL of the Flow Cell Wash Mix from step 81 into the priming port.
Do not add all of the mix. Keep some liquid in the pipette tip to avoid introducing air bubbles.
Close the flow cell priming port cover.
Wait for 60 min.
While you wait, thaw the reagents needed to prime the flow cell.
Perform steps 60 and 61 of the "Priming the flow cell" section to prepare the priming solutions.
Ensure that both the priming port cover and the SpotON sample port cover are closed.





Use a P1000 to remove all liquid waste from the Waste Port 1. See step 83.
Follow the steps (63–66) in the "Priming the flow cell" section to prime the flow cell.
During the 5 min incubation of the flow cell during the priming steps, warm the recovered DNA library to room temperature.
After the flow cell has been primed, open up the SpotON sample port.
Add ~Amount150 µL of the recovered room temperature DNA library to the sample port in a drop-wise manner.
Close the SpotON sample port.
Close the lid of the MinION device.
Restart the sequencing run on the MinKNOW software.
Protocol references
Dimitrios J. Camacho, Sabeeha Merchant 2024. Determination of Auxenochlorella protothecoides Cell Density With a Hemocytometer. protocols.iohttps://dx.doi.org/10.17504/protocols.io.rm7vzjk12lx1/v1

Frédéric Chaux-Jukic, Nicolas Agier, Stephan Eberhard, Zhou Xu 2024. Extraction and selection of high-molecular-weight DNA for long-read sequencing from Chlamydomonas reinhardtii . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv59j9jg1b/v2

Mukhopadhyay, T., & Roth, J. A. (1993). Silicone lubricant enhances recovery of nucleic acids after phenol-chloroform extraction. Nucleic acids research, 21 (3), 781. doi: 10.1093/nar/21.3.781

Acknowledgements
We thank Dr. Claire Remacle for help with troubleshooting and optimizing the high molecular weight DNA extraction and adapter ligation methods, Dr. Nelson Garcia, Sequencing Applications Scientist at Oxford Nanopore Technologies, for guidance and recommendations, and Dr. Sean Gallaher for technical assistance regarding the IT requirements and MinKNOW software operation.