TITLE: Nuclear DNA purification from recalcitrant plant species for long-read sequencing
AUTHORS: Ashley Jones, Justin Borevitz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Evolution has driven genetic diversity of life on Earth, but also created highly complex genomes that are difficult to sequence. Current draft genomes can have thousands to hundreds of thousands of contigs rather than chromosomes, containing incorrect assemblies, gaps and errors. With rapid advances in long-read 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 recalcitrant native Australian trees such as Eucalypts and Acacias. To resolve this, firstly we optimised a density gradient based nuclei extraction to remove cytoplasmic secondary metabolites, phenols and limit reads from high copy count plastid genomes. Secondly, we optimised a gentle high-molecular weight DNA extraction free of columns and high centrifugation, to limit DNA fragmentation. Finally, DNA was purified and size selected by gel electrophoresis. For sequencing, we adopted the portable MinION sequencer from Oxford Nanopore Technologies. Using these approaches, we can approximately obtain over 10 gigabases of sequencing from a single MinION revC flow cell and over 15 gigabases with new revD flow cells, up to 23 gigabases. This includes quality reads over 200 kb in length, average N50 values over 20 kb and some N50 values exceeding 45 kb. Such ultra-long reads assist the assembly of high quality genomes, from telomere to telomere.</div></div>

[STEPS]
?. [NUCLEI ISOLATION]
Add 2.5 mL of 100% Triton X-100, or 25 mL of 10% (final concentration 0.5%).
?. [NUCLEI ISOLATION]
Incubate mixture on an ice bath with gentle rocking for 30 min.
Use this time to clean the blender and space used for filtering. Cool the the centrifuge to 4°C.
?. [NUCLEI ISOLATION]
Centrifuge at 600 rcf and 4°C for 20 minutes.
?. [NUCLEI ISOLATION]
Weigh the Nalgene bottles with contents and lids to ensure they are equal for balancing purposes. Also ensure the Nalgene bottle has an intact o-ring to avoid leakage during centrifugation.
?. [NUCLEI ISOLATION]
Centrifuge at 600 rcf and 4°C for 30 minutes.
4,600 rpm on a Sorvall RC5C using rotor SLA-3000 (00).
?. [NUCLEI ISOLATION]
Discard the supernatant and add up to 450 mL of nuclei wash buffer per Nalgene bottle (save 100 mL).
?. [NUCLEI ISOLATION]
Gently resuspend the pellet using a large fine hair paint brush, soaked in freshly made pre-chilled nuclei wash buffer.
Ensure the bottles are balanced and have an o-ring as previously described.
?. [NUCLEI ISOLATION]
Discard supernatant and repeat with another ~450 mL of nuclei wash buffer. The pellet should become grey-white with no traces of green. If not, make more nuclei wash buffer and repeat the resuspending and washing steps.
2 L of nuclei wash buffer is suitable for x2 samples to be washed x2 times with 450 mL each, then a final resuspension with 100 mL each.
?. [NUCLEI ISOLATION]
After the final spin down, discard the supernatant and resuspend the pellet with 50 mL of nuclei wash buffer. Transfer evenly across two 50 mL Falcon tubes. Repeat the resuspension with another 50 mL of buffer and add to the two Falcon tubes.
?. [NUCLEI ISOLATION]
Centrifuge at 600 rcf and 4°C for 30 minutes.
?. [NUCLEI ISOLATION]
The nuclei pellets can now be stored -80°C.
?. [DNA EXTRACTION FROM NUCLEI]
Prepare 10 mL of fresh lysis buffer per sample. Ideally, pre-heat at 50-65°C.
The solution should be clear before use.
?. [DNA EXTRACTION FROM NUCLEI]
To a 3-5 mL nuclei pellet, add 10 mL of lysis buffer. Vortex vigorously for 1 min.
The SDS will be approximately 2% final concentration. Usually just one of the two Falcon tubes of a nuclei prep per sample are used.
?. [DNA EXTRACTION FROM NUCLEI]
Add RNAse A and Proteinase K to the solution. Mix by swirling and inverting.  ABC1EnzymeStockQuantity
(10 mL lysis buffer)2400 μg/mL RNAse A20 mg/mL200 μL3100 μg/mL Proteinase K20 mg/mL50 μL
ABC1EnzymeStockQuantity
(10 mL lysis buffer)2400 μg/mL RNAse A20 mg/mL200 μL3100 μg/mL Proteinase K20 mg/mL50 μL
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 temperatures15-70°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.
?. [DNA EXTRACTION FROM NUCLEI]
Incubate the samples at 50-60°C for 1 h, shaking at 400 - 900 rpm if possible.
Higher temperatures and longer incubations lead to DNA damage.
?. [DNA EXTRACTION FROM NUCLEI]
Add 1/3 volume (4-5 mL) of 5 M Potassium Acetate and mix by inverting to precipitate the proteins and the polysaccharides that will complex with SDS.
?. [DNA EXTRACTION FROM NUCLEI]
Incubate on ice (4°C) for 10 min (don’t rotate, DNA vulnerable).
?. [DNA EXTRACTION FROM NUCLEI]
Centrifuge at 5,000 rcf for 5 min at 4°C.
?. [DNA EXTRACTION FROM NUCLEI]
Transfer supernatant to a new tube (15 mL Falcon), centrifuge again at 5,000 rcf for 10 min at 4°C.
?. [DNA EXTRACTION FROM NUCLEI]
Transfer to a new tube and add 1.2x binding buffer. Ideally, split across x2 15 mL Falcon tubes.
?. [DNA EXTRACTION FROM NUCLEI]
Add 1 mL of 2% Sera-Mag beads (500 µL per Falcon tube if split).
Beads are in excess and could be reduced.
?. [DNA EXTRACTION FROM NUCLEI]
Mix by inverting the tube 20 times. Incubate with gentle agitation using a rotator or a shaker platform for 1 h at room temperature.
?. [DNA EXTRACTION FROM NUCLEI]
Place the tube in a magnetic rack for 30 min or more (until the solution becomes clear, can be over an hour).
?. [DNA EXTRACTION FROM NUCLEI]
Remove the supernatant without disturbing the beads.
?. [DNA EXTRACTION FROM NUCLEI]
Wash the beads by filling the tube with 70% ethanol, let beads settle if disturbed, and pour out ethanol.
?. [DNA EXTRACTION FROM NUCLEI]
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 EXTRACTION FROM NUCLEI]
Transfer the beads into a 2 mL Eppendorf tube. This can be done by taking a Falcon tube off the rack, adding 1 mL of ethanol to dislodge the beads, transfer suspension to an Eppendorf tube, repeat with another 1 mL. Repeat proces with the other Falcon tube into another Eppendorf tube. Place the 2 mL Eppendorf tubes on the magnetic rack, remove supernatant and repeat the process until all beads are in the same tube.
?. [DNA EXTRACTION FROM NUCLEI]
Remove all traces of ethanol and let the beads air dry for 1-4 min. Do not let the beads dry completely, they will crack and significantly reduce DNA recovery.
?. [DNA EXTRACTION FROM NUCLEI]
Using a wide-bore pipette tip, add 220 µL of nuclease-free water to the beads, gently resuspending. Gently tapping the tube is also suitable. Incubate at room temperature for at least 10 min.
Larger volume based on PippinHT input across x1 whole cassette.
?. [DNA EXTRACTION FROM NUCLEI]
Transfer 200-220 µL of supernatant (contains DNA) to a DNA LoBind eppendorf tube, avoiding any carry-over of beads.
?. [DNA EXTRACTION FROM NUCLEI]
Place the tubes in the magnetic rack for ~30 min. Let the solution become clear.
Highly concentrated DNA will take a long time. The tube can be left on the magnetic rack overnight in the fridge, or add more water.
?. [GEL PURIFICATION: PIPPIN PREP]
For some species, the DNA may be pure enough for sequencing at this point. However, for most recalcitrant plants, the DNA will still have impurities and DNA fragmentation is inevitable. Gel purification is an ideal solution to both problems.
?. [GEL PURIFICATION: PIPPIN PREP]
Using a PippinHT (Sage Science) or similar automated electrophoresis product, gel purify approximately 30 µg, following the manufacturer’s instructions. A 15 kb high pass separation is recommended, however if DNA is plentiful, 20 kb high pass is more 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 successfully overloaded to 3 µg per lane (perhaps more). The size selection will not be as precise, but is of no concern here.
Alternatively, a chloroform: isoamyl alcohol 24:1 clean-up can be performed to remove residual phenols and proteins. Other possibilities (untested), are digestion with proteinase K and another bead clean up. Also would be interesting to experiment with adding PVP to the binding buffer to further reduce polyphenols.
?. [GEL PURIFICATION: PIPPIN PREP]
After separation, wait at least 45 min (hours or overnight is suitable), to aid elution and recovery.
?. [GEL PURIFICATION: PIPPIN PREP]
Collect the contents of all elution wells into a 1.5 mL DNA LoBind Eppendorf tube (approx. 300 µL).
?. [GEL PURIFICATION: 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 DNA LoBind Eppendorf tube (another 300 µL, tube total is approx. 600 µL).
?. [GEL PURIFICATION: PIPPIN PREP]
Add 1.2x binding buffer (approx. 720 µL), and 100 µL of 2% Sera-Mag beads to the 1.5 mL DNA LoBind Eppendorf tube. Incubate for 5-10 min at room temperature.
?. [GEL PURIFICATION: PIPPIN PREP]
Remove the tube from the magnetic rack and elute with 60 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).
?. [SEQUENCING LIBRARY PREPARATION]
For sequencing, we adopted the portable MinION sequencer from Oxford Nanopore Technologies. There are two native genomic DNA library preparations available; a rapid transposase based method (SQK-RAD004) and a ligation based method (SQK-LSK109). Following the manufacturer’s instructions, prepare a library. Note that Oxford Nanopore recommends a mass of DNA optimised for 0.2 pmol (193 fmol). However, this is based on an average length of 8 kb. Therefore, the amount of input DNA needs to be adjusted. The following is recommended based on our sequencing experiences realtive to PippinHT size selection:  ABC1Fragment sizesRapid SQK-RAD004
Ligation SQK-LSK1092Amplicon or digest (~1 kb)Not recommended200 ng3No size selection (~8 kb)800 ng2,000 ng415 kb gel purification1,000 ng3,000 ng520 kb gel purification1,200 ng3,000 ng630 kb gel purificationNot recommended4,000 ng740 kb gel purificationNot recommended5,000 ng
ABC1Fragment sizesRapid SQK-RAD004
Ligation SQK-LSK1092Amplicon or digest (~1 kb)Not recommended200 ng3No size selection (~8 kb)800 ng2,000 ng415 kb gel purification1,000 ng3,000 ng520 kb gel purification1,200 ng3,000 ng630 kb gel purificationNot recommended4,000 ng740 kb gel purificationNot recommended5,000 ng
?. Expected ResultsUsing the protocol described, we have been obtaining clean high molecular weight DNA (Figure 1, Table 1). DNA fragment size ranges from 20-200 kb in length (Figure 2). During sequencing, we can approximately obtain over 10 gigabases of sequencing from a single MinION revC flow cell, and over 15 gigabases with new revD flow cells, up to 23 gigabases (Table 2, Figure 3, Figure 4). This includes quality reads over 200 kb in length. N50 values depend on library preparation method, but can reproducibly obtain N50 values > 20 kb using ligation kits, and can exceed N50 of 45 kb. A troubleshooting guide is presented in Figure 5, illustrating the most common problems researchers have.
?. [NUCLEI ISOLATION]
Transfer supernatant into new 50 mL Falcon tubes and centrifuge at 1,000 rcf and 4°C for 30 minutes.
Optional. Recovers a small pellet that can be added to the larger pellet or used for other applications like HiC library preps.
?. [DNA EXTRACTION FROM NUCLEI]
Add an additional 100 µL of nuclease-free water to the beads, remove from magnet, resuspend, incubate, magnetise and transfer to a new 1.5 mL Eppendorf tube.
Save this second elution for other applications such as running a pulse field gel.
?. [GEL PURIFICATION: PIPPIN PREP]
Place on a magnetic rack for 5 min, or until the solution becomes clear. Discard the supernatant.
?. [GEL PURIFICATION: 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.
?. [GEL PURIFICATION: PIPPIN PREP]
Remove all traces of ethanol and let the beads air dry for 1-4 min. Do not let the beads dry completely, they will crack and significantly reduce DNA recovery.
?. [GEL PURIFICATION: 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 DNA LoBind Eppendorf tube using a wide-bore pipette tip.
?. [GEL PURIFICATION: 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 quantifications are 1:1.Expect 20-30% recovery relative to total input (~6-9 µg out of 30 µg).For pure DNA, Nanodrop: Qubit quantifications are 1:1.Expect 20-30% recovery relative to total input (~6-9 µg out of 30 µg).
?. [DNA EXTRACTION FROM NUCLEI]
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. However, as the DNA is crude, it will give a large over-estimation of concentreation.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.Expect 20-100 µg of DNA.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.Expect 20-100 µg of DNA.
?. [GEL PURIFICATION: PIPPIN PREP]
Add an additional 60 µL of nuclease-free water to the beads, remove from magnet, resuspend, incubate, magnetise and transfer to a new 1.5 mL Eppendorf tube.
Save this second elution for other applications such as running a pulse field gel.
?. [SEQUENCING LIBRARY PREPARATION]
Load the MinION and perform sequencing according to Oxford Nanopore's instructions. Ensure no air bubbles are introduced into the array during loading.
Air bubbles destroy nanopores, significantly reducing sequencing yield.
?. [DNA EXTRACTION FROM NUCLEI]
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 3 months.
?. [GEL PURIFICATION: 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 3 months.
?. [GEL PURIFICATION: 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.
?. [SKIPPING NUCLEI: ALTERNATIVE LYSIS (optimistic)]
For soft tissue with with a high cell density, approximately 1 g (0.5-1.5 g) of tissue can be ground in a mortar and pestle, keeping frozen with liquid nitrogen. Then proceed to 'DNA EXTRACTION FROM NUCLEI', using the ground tissue instead of a nuclei pellet. For recalcitrant species, purify nuclei as detailed below from 'BLENDER HOMOGENISATION'.
This has been trialled with wild and cultivated rice, being hilghly successful (400 and 900 Mb genomes). Eucalyptus will not have a sufficient yield and will be highly contaminated.
?. [BLENDER HOMOGENISATION (4°C cold room)]
Place 50 g of leaves into a blender with enough nuclei isolation buffer to cover the blades (approximately 150 mL).
Optimised for Eucalyptus, a diploid ~500 Mb genome. Larger genomes or polyploid plants can have less input accordingly.
?. [BLENDER HOMOGENISATION (4°C cold room)]
Homogenise the tissue on max speed. Transfer to a beaker, repeatedly washing out blender with nuclei isolation buffer (500 mL buffer is allocated per extraction).
?. [BLENDER HOMOGENISATION (4°C cold room)]
Clean the blender with tap water, 70% ethanol and then MQW; process the other sample (repeating steps 1 and 2).
?. [FILTRATION (ideally 4°C cold room)]
Filter homogenate using a sieve (or disk membrane mesh placed in a funnel) into a 1 L Schott bottle. Forcibly squeeze out as much residual homogenate from the leaf debris as possible, maximising nuclei capture.
?. [FILTRATION (ideally 4°C cold room)]
Filter homogenate through 1 layer of Miracloth using a funnel and 1 L Schott bottle. Gently squeeze residual homogenate through the Miracloth.
May need to change the layer half way through (very dirty and clogged).
?. [FILTRATION (ideally 4°C cold room)]
Repeat Filtration through 2 layers of Miracloth then 4 layers (using new Schott bottles).
?. [FILTRATION (ideally 4°C cold room)]
Finally, gravity filter through 8 layers of Miracloth (no squeezing) into a 500 mL Nalgene bottle. Repeat steps 4-6 for the second sample.