TITLE: Scalable high-molecular weight DNA extraction for long-read sequencing
AUTHORS: Ashley Jones, Cynthia Torkel, David Stanley, Jamila Nasim, Justin Borevitz, Benjamin Schwessinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>With rapid advances in long-read DNA sequencing technologies, it is becoming possible to resolve complex genomes, including repetitive, polyploid plant genomes. Despite the technology being available, a challenge persists: the extraction of pure high molecular weight DNA suitable for long-read sequencing. This is particularly true of native plants, crops and fungi. To resolve this, we optimised a gentle magnetic bead based high-molecular weight DNA extraction free of columns and high-centrifugation, to limit DNA fragmentation. A protocol that is scalable based on tissue input is presented, that can be used on many species of plants, fungi, reptiles, insects and bacteria. An optional sorbitol wash is listed and is highly recommended for plant tissues. To remove any remaining contaminants such as phenols and polysaccharides, two optional DNA clean-up and size selection strategies are given. Sequencing with Oxford Nanopore Technologies MinION, we can approximately obtain over 15-30 Gbp of sequencing from a single MinION flow cell with N50 values 30-50 kb. This has been routinely achieved with eucalypts, acacias, rice, themeda, wheat, wheat rusts, various other fungi, geckos, skinks, ticks, ladybird beetles, caterpillars and </span><span style = "font-style:italic;">E. coli</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
?. [PREPARATION]
Set a water bath (or equivalent alternative) to 55°C (temperature range 50-60°C)
Will be used to dissolve lysis buffer and also used during lysis (30-90 min).
?. [PREPARATION]
Prepare fresh lysis buffer based on the prep size and heat at 55°C in water bath until ready to use.  ABC1Prep sizeTissue inputAmount needed per sample2Small (2 mL tube prep)100-200 mg750 μL3Medium (50 mL Falcon tube prep)1-3 g20 mL4Large (250 mL Schott bottle prep)≤ 30 g100 mL  ABCDE1ReagentTarget concentrationMWStockFrom stock (10 mL)2Polyvinylpyrrolidone 40 (PVP-40)1%40,00010%
1 mL
3Sodium metabisulfite1%190.1110%
1 mL
4Sodium chloride (NaCl)0.5 M58.445 M
1 mL
5TRIS-HCl pH 8.0100 mM121.141 M
1 mL
6EDTA pH 8.050 mM292.240.5 M
1 mL
7Sodium dodecyl sulfate (SDS)2%288.3720%
1 mL8Water (Milli-Q)NA--
4 mL9EGTA pH 8 (optional, see below)6 mM380.350.5 M120 μL
ABC1Prep sizeTissue inputAmount needed per sample2Small (2 mL tube prep)100-200 mg750 μL3Medium (50 mL Falcon tube prep)1-3 g20 mL4Large (250 mL Schott bottle prep)≤ 30 g100 mL
ABCDE1ReagentTarget concentrationMWStockFrom stock (10 mL)2Polyvinylpyrrolidone 40 (PVP-40)1%40,00010%
1 mL
3Sodium metabisulfite1%190.1110%
1 mL
4Sodium chloride (NaCl)0.5 M58.445 M
1 mL
5TRIS-HCl pH 8.0100 mM121.141 M
1 mL
6EDTA pH 8.050 mM292.240.5 M
1 mL
7Sodium dodecyl sulfate (SDS)2%288.3720%
1 mL8Water (Milli-Q)NA--
4 mL9EGTA pH 8 (optional, see below)6 mM380.350.5 M120 μL
Prepare lysis buffer fresh on the day of use for optimal results.The solution should be clear before use. EGTA is recommended for wheat and very soft plant tissues that wilt fast and/or have high amounts of endogenous DNases.EDTA has a high-affinity for Mg2+, EGTA has a high-affinity for Ca2+. Prokaryotic DNases are dependent on Mg2+, plant DNAses Ca2+.
?. [PREPARATION]
Ensure there is enough 5 M potassium acetate, binding buffer and Sera-Mag beads to carry out the prep size and quantity (listed in Materials).
?. [HOMOGENISATION]
[Bacteria cultures only] Pellet cells in a 2 mL tube or 50 mL Falcon tube by centrifuging at 5,000 rcf for 10 min at room temperature and discard growth medium. Skip next step (grinding) and proceed to adding lysis buffer.
?. [HOMOGENISATION]
Grind tissue to a fine powder, keeping frozen with liquid nitrogen.   ABC1Prep sizeTissue inputRecommendation2Small (2 mL tube prep)100-200 mg1-3 ball bearings in 2 mL tube, place in TissueLyser (Qiagen) for 2 min at 25 Hz.3Medium (50 mL Falcon tube prep)1-3 gMortar and pestle, transfer to a 50 mL Falcon tube.4Large (250 mL Schott bottle prep)≤ 30 gMortar and pestle, or blender. Transfer to 250 mL Schott bottle.
ABC1Prep sizeTissue inputRecommendation2Small (2 mL tube prep)100-200 mg1-3 ball bearings in 2 mL tube, place in TissueLyser (Qiagen) for 2 min at 25 Hz.3Medium (50 mL Falcon tube prep)1-3 gMortar and pestle, transfer to a 50 mL Falcon tube.4Large (250 mL Schott bottle prep)≤ 30 gMortar and pestle, or blender. Transfer to 250 mL Schott bottle.
?. [SORBITOL WASH (OPTIONAL)]
[Optional] If the sample is likely to have a high amount of sugars, oils and/or other endogenous chemicals present (e.g. plant tissue), perform a sorbitol wash. See the following protocol:Sorbitol washing complex homogenate for improved DNA extractionsJones, A., and Schwessinger, B. (2020). Sorbitol washing complex homogenate for improved DNA extractions. Protocols.io v1, dx.doi.org/10.17504/protocols.io.beuvjew17506.
?. [CELL LYSIS]
Add lysis buffer to tissue. Vortex or shake vigorously to mix.  AB1Prep sizeLysis buffer to add2Small (2 mL tube prep)750 μL3Medium (50 mL Falcon tube prep)Bring volume to 20 mL4Large (250 mL Schott bottle prep)Bring volume to 100 mL
AB1Prep sizeLysis buffer to add2Small (2 mL tube prep)750 μL3Medium (50 mL Falcon tube prep)Bring volume to 20 mL4Large (250 mL Schott bottle prep)Bring volume to 100 mL
Lysis buffer should be 1-2x the approximate volume of the ground tissue.
?. [CELL LYSIS]
[Bacteria cultures only] Add a small scoop of lysozyme to the lysis reaction, approximately 2-4 mg/mL.
?. [CELL LYSIS]
Add RNAse A and Proteinase K to the solution. Mix by swirling and inverting.[Bacteria cultures only] Do not add Proteinase K yet.   ABCD1Prep sizeLysis volumeRNAse A (20 mg/mL stock)Proteinase K (20 mg/mL stock)2Small (2 mL tube prep)750 μL8 μL (213 μg/mL final)4 μL (107 μg/mL final)3Medium (Falcon tube prep)20 mL200 μL (200 μg/mL final)100 μL (100 μg/mL final)4Large (Schott bottle prep)100 mL400 μL (80 μg/mL final)200 μL (40 μg/mL final)
ABCD1Prep sizeLysis volumeRNAse A (20 mg/mL stock)Proteinase K (20 mg/mL stock)2Small (2 mL tube prep)750 μL8 μL (213 μg/mL final)4 μL (107 μg/mL final)3Medium (Falcon tube prep)20 mL200 μL (200 μg/mL final)100 μL (100 μg/mL final)4Large (Schott bottle prep)100 mL400 μL (80 μg/mL final)200 μL (40 μg/mL final)
Proteinase K and RNAse A can co-exist in the same solution, even in the presence of EDTA.RNAse A is highly resistant to proteolysis by Proteinase K.Activity of RNAse A and Proteinase K is not dependent on ion cofactors, EDTA has no effect.Both are recommended to be 50-100 µg/mL.Qiagen DNeasy plant kits use as much as 2,000 μg/mL RNAse A.RNAse T has less DNase activity (still present), but was less effective at degrading RNA.RNAse A is active at temperatures 15-70°C (optimal at 60°C), pH 6-10 (optimal pH 7.6).Proteinase K is active at temperatures 20-60°C (optimal 50-60°C), pH 4-12 (optimal pH 8).Proteinase K degrades proteins in the presence of detergents, including SDS.Proteinase K activity is stimulated if up to 2% SDS or 4 M urea is present in the reaction.Lysozyme activity varies based on where it was purified from. Generally, active at temperatures 20-55°C (optimal at 30-50°C), pH 6-9 (optimal pH 6.2).
?. [CELL LYSIS]
Incubate the samples at 55°C, shaking at ~450 rpm if possible. Recommended time:  AB1Prep sizeIncubation time2Small (2 mL tube prep)60 min3Medium (50 mL Falcon tube prep)60 min4Large (250 mL Schott bottle prep)90-120 min5[Bacteria cultures only]30 min
AB1Prep sizeIncubation time2Small (2 mL tube prep)60 min3Medium (50 mL Falcon tube prep)60 min4Large (250 mL Schott bottle prep)90-120 min5[Bacteria cultures only]30 min
Higher temperatures and longer incubations lead to DNA damage.
?. [CELL LYSIS]
[Bacteria cultures only] Add Proteinase K (see previous table) and incubate at 55°C for an additional 30 min.
?. [CONTAMINANT REMOVAL]
[Large prep only] Filter the homogenate using a sieve (or disk membrane mesh placed in a funnel) into a new 250 mL Schott bottle. Forcibly squeeze out as much residual homogenate from the debris as possible. Then split the homogenate into 50 mL Falcon tubes, bringing the volume to 20 mL for each.
?. [CONTAMINANT REMOVAL]
Add 1/3 volume of 5 M Potassium Acetate and mix by inverting to precipitate the proteins and the polysaccharides that will complex with SDS.   AB1Prep sizePotassium acetate2Small (2 mL tube prep)~250 μL3Medium (50 mL Falcon tube prep)~7 mL4Large (250 mL Schott bottle prep)~7 mL per 50 mL Falcon tube
AB1Prep sizePotassium acetate2Small (2 mL tube prep)~250 μL3Medium (50 mL Falcon tube prep)~7 mL4Large (250 mL Schott bottle prep)~7 mL per 50 mL Falcon tube
?. [CONTAMINANT REMOVAL]
Incubate on ice (4°C) for 10 min (don’t rotate, DNA vulnerable).
?. [CONTAMINANT REMOVAL]
Centrifuge at 5,000 rcf for 5 min at 4°C.
?. [CONTAMINANT REMOVAL]
Transfer supernatant to a new tube, centrifuge again at 5,000 rcf for 10 min at 4°C.
?. [CONTAMINANT REMOVAL]
Transfer supernatant to a new tube.
?. [DNA BINDING AND WASH]
Add approximately an equal volume of binding buffer or more. Utilise the capacity of the tube, leaving only a small space for adding Sera-Mag beads. Approximate amounts:  ABC1Prep sizeSample volumeBinding buffer2Small (2 mL tube prep)~950 μLBring volume to 1.90 mL3Medium (50 mL Falcon tube prep)~25 mLBring volume to 50 mL4Large (250 mL Schott bottle prep)~25 mL per 50 mL Falcon tubeBring volume to 50 mL
ABC1Prep sizeSample volumeBinding buffer2Small (2 mL tube prep)~950 μLBring volume to 1.90 mL3Medium (50 mL Falcon tube prep)~25 mLBring volume to 50 mL4Large (250 mL Schott bottle prep)~25 mL per 50 mL Falcon tubeBring volume to 50 mL
Adding more buffer can increase to yield, especially if the sample has a lot of contaminants. More shorter fragments will also be recovered, but can be size selected against later.
?. [DNA BINDING AND WASH]
Thoroughly mix the 2% Sera-Mag beads and add them to the sample:  AB1Prep sizeSera-Mag beads2Small (2 mL tube prep)100 μL3Medium (50 mL Falcon tube prep)1 mL4Large (250 mL Schott bottle prep)1 mL per Falcon or 10 mL overall
AB1Prep sizeSera-Mag beads2Small (2 mL tube prep)100 μL3Medium (50 mL Falcon tube prep)1 mL4Large (250 mL Schott bottle prep)1 mL per Falcon or 10 mL overall
?. [DNA BINDING AND WASH]
Mix by inverting the tube 20 times. Incubate with gentle mixing using a rotator or a shaker platform at room temperature.  AB1Prep sizeRecommended time2Small (2 mL tube prep)≥ 10 min3Medium (50 mL Falcon tube prep)≥ 60 min or overnight4Large (250 mL Schott bottle prep)≥ 120 min or overnight
AB1Prep sizeRecommended time2Small (2 mL tube prep)≥ 10 min3Medium (50 mL Falcon tube prep)≥ 60 min or overnight4Large (250 mL Schott bottle prep)≥ 120 min or overnight
?. [DNA BINDING AND WASH]
Place the tube in a magnetic rack until the solution becomes clear.  AB1Prep sizeRecommended time2Small (2 mL tube prep)≥ 10 min3Medium (50 mL Falcon tube prep)≥ 30 min4Large (250 mL Schott bottle prep)≥ 120 min or overnight
AB1Prep sizeRecommended time2Small (2 mL tube prep)≥ 10 min3Medium (50 mL Falcon tube prep)≥ 30 min4Large (250 mL Schott bottle prep)≥ 120 min or overnight
?. [DNA BINDING AND WASH]
Remove the supernatant without disturbing the beads, keeping the tube on the magnetic rack.
Small preps can be done by pipetting, larger preps by decanting.
?. [DNA BINDING AND WASH]
Wash the beads by filling the tube with 70% ethanol, let beads settle if disturbed, and decant out the ethanol.
?. [DNA BINDING AND WASH]
Repeat the ethanol wash another 2 times, or until satisfied the beads are clean.
If the beads are very dirty, remove the tube from the magnetic rack, resuspend beads by flicking the tube, magnetise until clear, remove supernatant, repeat washing.
?. [DNA BINDING AND WASH]
For medium and large preps, transfer the beads into a 2 mL Eppendorf tube. This can be done by taking the Falcon tube off the rack, adding 1 mL of 70% ethanol to dislodge the beads, transfer suspension to an Eppendorf tube. Repeat with another 1 mL to ensure all beads are transferred. Place the 2 mL Eppendorf tube on the magnetic rack, remove supernatant and repeat the process with other Falcon tubes until all beads are in the same eppendorf tube.
Do not let the beads dry during the process, keep the beads wet with ethanol and perform the process quickly. Larger preps can be done at the sink to discard ethanol easily.
?. [DNA BINDING AND WASH]
Remove all traces of ethanol and let the beads air dry for 1-4 min.
Important! Do not let the beads dry completely, they will crack and significantly reduce DNA recovery.
?. [DNA BINDING AND WASH]
Remove tube from magnetic rack and add 10 mM TRIS-HCl pH 8 (or nuclease-free water) to the beads, gently resuspending (use wide-bore pipette tip if available). Gently tapping the tube is also suitable.If the solution is very thick and cloudy white, there is a large yield; consider slowly increasing the elution volume (in some cases, double or triple the volume, e.g. large genomes, high cell density).  AB1Prep sizeRecommended elution2Small (2 mL tube prep)50 µL3Medium (50 mL Falcon tube prep)200 µL4Large (250 mL Schott bottle prep)500 µL
AB1Prep sizeRecommended elution2Small (2 mL tube prep)50 µL3Medium (50 mL Falcon tube prep)200 µL4Large (250 mL Schott bottle prep)500 µL
Larger volume is based on size selection protocols, e.g. PippinHT input across x1 whole cassette or a DNA clean-up protocol.
?. [DNA BINDING AND WASH]
Incubate at room temperature for at least 10 min.
?. [DNA BINDING AND WASH]
Place the tubes in the magnetic rack until the solution becomes clear.
Highly concentrated DNA will take a long time. The tube can be left on the magnetic rack overnight in the fridge, or increase the elution volume.
?. [DNA BINDING AND WASH]
Transfer the supernatant (contains DNA) to a 1.5 mL Eppendorf tube, avoiding any carry-over of beads. Use DNA LoBind tube if available.
?. [DNA BINDING AND WASH]
Perform an additional, second elution on the beads (same volume as previous), remove from magnet, resuspend, incubate, magnetise and transfer to another 1.5 mL Eppendorf tube. Use DNA LoBind tube if available.
Save this second elution for other applications such as running a pulse field gel.
?. [DNA BINDING AND WASH]
Quantify the DNA on a Nanodrop and a Qubit fluorometer (dsDNA broad-range assay) (both instruments from Thermo Fisher Scientific). The DNA is still crude and will likely have other contaminants present.
For Nanodrop, use 1 μL. As the DNA is crude, it may give a large over-estimation of concentration.Qubit fluorometer is the most reliable. However, it is highly dependent on the accuracy of the amount pipetted. Use 2 μL when sample is plentiful. Using 1 μL is prone to pipetting errors.For pure DNA, Nanodrop:Qubit is 1:1, 260/280 is 1.8 and 260/230 is 2.0.DNA yield can be 20-500 µg.
?. [DNA BINDING AND WASH]
Store DNA at 4°C to prevent cycles of freeze-thawing that shear the DNA.
No effects on DNA integrity have been noticed for samples stored at 4°C for 12 months.
?. [DNA BINDING AND WASH]
For some species, the DNA may be pure enough to proceed to sequencing. If the DNA is crude (likely for plant samples), consider DNA clean-up and size slection options below.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 1: SHORT-READ ELIMINATOR]
If the DNA is clear, has small quantities of impurities, consider utilising a Short-Read Elimator kit from Circulomics. If the DNA is discoloured with visbible impurities, it is best for perform a DNA clean-up first before using the Short-Read Elimator kit. See the following protocol:DNA clean-up and size selection for long-read sequencingJones, A., Purushotham, N., Nasim, J., and Schwessinger, B. (2020). DNA clean-up and size selection for long-read sequencing. Protocols.io v3, dx.doi.org/10.17504/protocols.io.betdjei17506.
It has been observed that the Short-Read Eliminator kit can result in and unexpected increased absorbance/fluorescence from Nanodrop/Qubit results. If this is seen, consider using more DNA input into the sequencing library prep than recommended. Perhaps the DNA double helix becomes altered or unwound. Alternatively, what is traditionally measured as DNA concentration has always been an under-estimate.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
For most recalcitrant plants, the DNA will still have a high quantity of impurities and DNA fragmentation is inevitable. Gel purification is an ideal solution to both problems. Proceed to gel purification by Pippin Prep (Sage Science) or equivalent.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
If a precipitant is noticebale in the DNA solution, briefly spin down and take the upper aqueous layer to gel purification below.
For some samples, this may be carbohydrates, which will be removed during gel purification. DNA is in the aqueous layer. It is possible 4°C promotes this precipitation.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Using a PippinHT (Sage Science) or similar automated electrophoresis product, gel purify approximately 30 µg, following the manufacturer’s instructions. A 20 kb high pass separation is recommended, however if DNA is limited, 15 kb high pass is suitable. The PippinHT has 12 lanes, however a lane pair needs be dedicated to an external ladder, leaving 10 lanes for samples. 20 µL of DNA goes into each lane (therefore 200 µL elution in previous section). The manufacturer recommends a maximum 1.5 µg per lane (15 µg total per cassette), however, can be safely overloaded to 3 µg per lane (30 µg total) without noticable consequences. Overloading further slows the migration of longer fragments and shorter fragments are eluted as the size selection will not be precise.
If the sample if very dirty and may not electrophorese correctly, consider a chloroform: isoamyl alcohol 24:1 clean-up before using the PippinHT.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
After separation, wait at least 45 min (hours or overnight is suitable), to aid elution and recovery.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Collect the contents of all elution wells into a 1.5 mL Eppendorf tube (approx. 300 µL). Use DNA LoBind tube if available.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Add 30 µL of 0.1% tween in electrophoresis buffer to each elution well (provided in kit). Wait for 5 min and then transfer the contents to the same 1.5 mL Eppendorf tube (another 300 µL, tube total is approx. 600 µL).
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Add 1.2x binding buffer (approx. 720 µL), and 100 µL of 2% Sera-Mag beads to the 1.5 mL Eppendorf tube. Incubate for 5-10 min at room temperature.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Place on a magnetic rack for 5 min, or until the solution becomes clear. Discard the supernatant.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Keeping the tube on the magnetic rack, add 1 mL of freshly prepared 70% ethanol. Discard the ethanol and repeat for a second ethanol wash.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Remove all traces of ethanol and let the beads air dry for 1-4 min.
Important! Do not let the beads dry completely, they will crack and significantly reduce DNA recovery.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Remove the tube from the magnetic rack and elute with 50 µL of nuclease-free water. Incubate for 10 min at room temperature.
A maximum DNA volume of 48 µL is used in an Oxford Nanopore ligation prep (e.g. SQK-LSK109).
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Place on a magnetic rack for 5 min, or until the solution becomes clear. Transfer eluted DNA to a new 1.5 mL Eppendorf tube. Use DNA LoBind tube if available. Use a wide-bore pipette tip if available.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Perform an additional, second elution on the beads (same volume as previous), remove from magnet, resuspend, incubate, magnetise and transfer to another 1.5 mL Eppendorf tube. Use DNA LoBind tube if available.
Save this second elution for other applications such as running a pulse field gel.
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Quantify the DNA on a Nanodrop and a Qubit fluorometer (dsDNA broad-range assay) (both instruments from Thermo Fisher Scientific). The DNA should be pure, free of contaminants.
For Nanodrop, use 1 μL.Qubit fluorometer is highly dependent on the accuracy of the amount pipetted. Use 2 μL when sample is plentiful. Using 1 μL is prone to pipetting errors.For pure DNA, Nanodrop:Qubit is 1:1, 260/280 is 1.8 and 260/230 is 2.0.Expect 20-30% recovery relative to total input (~6-9 µg out of 30 µg).
?. [DNA CLEAN-UP AND SIZE SELECTION OPTION 2: GEL PURIFICATION BY PIPPIN PREP]
Store DNA at 4°C to prevent cycles of freeze-thawing that shear the DNA.
No effects on DNA integrity have been noticed for samples stored at 4°C for 12 months.