TITLE: High-molecular weight DNA extraction and small fragment removal of Ascochyta lentis
AUTHORS: Johannes Wolfram Debler, Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>Extracting pure high-molecular weight DNA from some fungal species is difficult due to the presence of polysaccharides and potentially other compounds which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, being difficult to separate fom the DNA. Although the contaminant may not be detected by spectrophotometers or fluorometric devices, it substantially interferes with long-read DNA sequencing, such as Oxford Nanopore Technologies. To partially resolve this, a protocol is presented with some updates to current strategies and incorporates a small fragment removal step using Polyethylene Glycol. Using this protocol, we have been successfully sequencing the lentil pathogen </span><span style = "font-style:italic;">Ascochyta lentis</span><span style = "font-style:italic;font-style:italic;"> </span><span style = "font-style:italic;"> </span><span>with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 13 gigabases with an N50 of approximately 15 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants. </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>DNA extraction modified from: </span><a href="https://www.protocols.io/view/high-molecular-weight-dna-extraction-from-challeng-5isg4ee" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/high-molecular-weight-dna-extraction-from-challeng-5isg4ee</span></a></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span>PEG small fragment elimination after: </span><a href="https://www.protocols.io/view/size-selective-precipitation-of-dna-using-peg-amp-7erhjd6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/size-selective-precipitation-of-dna-using-peg-amp-7erhjd6</span></a></div></div></div>

[STEPS]
?. [PREPARATION]
Prepare about 500 mg of fungal mycelia grown in liquid media (YEG, 0.5% Yeast Extract, 5% Glucose). Remove media by filtering through a sterile milk filter disk. Wash mycelia with Milli-Q water while on the filter and keep frozen in liquid nitrogen.
?. [PREPARATION]
Set a water bath to 55°C. This will be used to dissolve 2% CTAB solutions and to preciptate DNA with CTAB.
?. [PREPARATION]
Freshly prepare lysis and precipitation buffers.Lysis buffer5 mL per 500 mg of sample.  Precipitation buffer10 mL per 500 mg of sample.    9% PEG8000 small fragment elimination buffer~300 uL per 500 mg of sample.  ABCD1ComponentMWStockQuantity
(5 mL)22% CTAB (w/v)364.45powder0.10 g3100 mM Tris-HCl (pH 8)157.601 M0.50 mL420 mM EDTA (pH 8)292.240.5 M0.20 mL51.2 M NaCl58.445 M1.20 mL61% PVP40,00010%0.50 mL7Milli-Q water--2.60 mLPrecipitation buffer10 mL per 500 mg of sample.    9% PEG8000 small fragment elimination buffer~300 uL per 500 mg of sample.  ABCD1ComponentMWStockQuantity
(10 mL)22% CTAB (w/v)364.45powder0.20 g3100 mM Tris-HCl (pH 8)157.601 M1 mL420 mM EDTA (pH 8)292.240.5 M0.40 mL5Milli-Q water--8.60 mL9% PEG8000 small fragment elimination buffer  ABCD1ComponentMWStockQuantity (10 mL)29% PEG 8000800018%5 mL31M NaCl58.445 M2 mL410 mM Tris-Cl (pH 8.0)157.601 M100 uL5Milli-Q water--2.9 mL
ABCD1ComponentMWStockQuantity
(5 mL)22% CTAB (w/v)364.45powder0.10 g3100 mM Tris-HCl (pH 8)157.601 M0.50 mL420 mM EDTA (pH 8)292.240.5 M0.20 mL51.2 M NaCl58.445 M1.20 mL61% PVP40,00010%0.50 mL7Milli-Q water--2.60 mL
ABCD1ComponentMWStockQuantity
(10 mL)22% CTAB (w/v)364.45powder0.20 g3100 mM Tris-HCl (pH 8)157.601 M1 mL420 mM EDTA (pH 8)292.240.5 M0.40 mL5Milli-Q water--8.60 mL
The DNA extraction process appeared unaffected when the lysis and precipitation buffers were not adjusted to pH 8. Note Tris-HCl and EDTA stock solutions are already at pH 8.
ABCD1ComponentMWStockQuantity (10 mL)29% PEG 8000800018%5 mL31M NaCl58.445 M2 mL410 mM Tris-Cl (pH 8.0)157.601 M100 uL5Milli-Q water--2.9 mL
?. [PREPARATION]
Vortex lysis and precipitation buffers, then place both in the 55°C water bath to dissolve CTAB. Further vortexing or inverting may be needed to dissolve all CTAB. Leave buffers at 55°C until needed.
?. [SAMPLE LYSIS]
Place a sterile mortar and pestle into an insulated container. Pour liquid nitrogen into the container to chill the mortar and pestle.
?. [SAMPLE LYSIS]
Add mycelium to the mortar and grind for approximately 1-2 min. Keep the mortar and pestle chilled by having liquid nitrogen within the surrounding container.
?. [SAMPLE LYSIS]
Transfer the ground mycelium to 5 mL lysis buffer in a 15 mL falcon tube.
?. [SAMPLE LYSIS]
Add RNAse A and Proteinase K to the solution:  ABC1EnzymeStockQuantity
(5 mL lysis buffer)2100 μg/mL RNAse A10 mg/mL50 μL3100 μg/mL Proteinase K20 mg/mL25 μL
ABC1EnzymeStockQuantity
(5 mL lysis buffer)2100 μg/mL RNAse A10 mg/mL50 μL3100 μg/mL Proteinase K20 mg/mL25 μL
RNAse A was added earlier than previous protocols and concentration doubled as RNA has been still present after extraction. Athough RNAse A concentration is generally reccomended to be 1-100 μg/mL, Qiagen DNeasy plant kits use as much as 2,000 μg/mL. RNAse T has less DNase activity (still present), but was less effective at degrading fungal RNA.RNAse A activity is not Mg2+ dependent and doesn't appear to require metal ions as cofactors. Similarily, Proteinase K is still active in EDTA, as the two binding sites for Ca2+ are not directly involved in the proteolysis catalytic mechanism.Proteinase K and RNAse A can co-exist in the same solution. RNAse A is highly resistant to proteolysis by Proteinase K. Both are recommended to be 50-100 µg/mL. 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).RNAse A activity is not Mg2+ dependent and doesn't appear to require metal ions as cofactors. Similarily, Proteinase K is still active in EDTA, as the two binding sites for Ca2+ are not directly involved in the proteolysis catalytic mechanism.Proteinase K and RNAse A can co-exist in the same solution. RNAse A is highly resistant to proteolysis by Proteinase K. Both are recommended to be 50-100 µg/mL. 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).
?. [SAMPLE LYSIS]
Vortex and invert the solution. Incubate at 55°C for at least 1 h.
?. [CHLOROFORM CLEAN-UP]
Add an equal volume of chloroform: isoamyl alcohol (24:1, v/v) and mix by inverting 10-15 times. Ensure the organic and aqueous phases become mixed at least temporarily.
?. [CHLOROFORM CLEAN-UP]
Separate the phases by centrifuging at 5,000 rcf for 10 min at 20°C. Transfer the upper aqueous phase to a new 15 mL Falcon tube.
Chloroform is denser than water, will mix with the oragnic phase which settles to the bottom and the aqueous phase is at the top. Some protocols recommend centrifuging at 12,000 rpm. Not tested, however if centrifugation hasn't been sufficient the interphase will be cloud-like and poorly compacted, with oraganic substances still in the aqueous phase.
?. [CHLOROFORM CLEAN-UP]
Repeat the chloroform: isoamyl alcohol clean (equal volume).
?. [DNA PRECIPITATION]
Add 2 volumes of precipitation buffer, mix by inverting. Incubate at 55°C for 1 h or until white crystals of CTAB-DNA complex can be observed floating inside the tubes.
?. [DNA PRECIPITATION]
Centrifuge at 5,000 rcf for 30 minutes at 20°C to pellet crystals.
?. [DNA PRECIPITATION]
Carefully decant the supernatant as soon as possible, without disturbing the pellet. Care must be taken as the pellet is fragile, being easily dislodged.
?. [DNA PRECIPITATION]
Add enough freshly prepared 70% ethanol to cover the pellet (~5 mL). Let the pellet soak for 15 min at room temperature to dissolve excess salts and CTAB.
?. [DNA PRECIPITATION]
Centrifuge at 5,000 rcf for 10 min. Carefully decant the supernatant as soon as possible, without disturbing the pellet.
?. [DNA PRECIPITATION]
Air-dry the pellet for 10-15 min, or until all ethanol has evaporated.
?. [DNA PRECIPITATION]
Dissolve DNA with a maximum of 300 µL in 0.1x TE in nuclease-free water. Avoid pipette mixing as much as possible to prevent DNA shearing. Gentle flicking of the tube and incubating at room temperature is ideal. Wide-bore pipette tips are also an option.
Volume is based on loading the sample across a whole PippinHT (Sage Science) casette at later steps.If planning to deviate from this protocol and perform other clean-ups or enzymatic digestions, it is recommended to dissolve the pellet in 50-500 μL of 10 mM Tris (pH 8.0). This keeps the DNA buffered and in solution.This step can take a couple of days!If planning to deviate from this protocol and perform other clean-ups or enzymatic digestions, it is recommended to dissolve the pellet in 50-500 μL of 10 mM Tris (pH 8.0). This keeps the DNA buffered and in solution.This step can take a couple of days!
?. [DNA PRECIPITATION]
Transfer DNA to a 1.5 mL DNA LoBind Eppendorf tube.
?. [DNA PRECIPITATION]
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 RNA and other contaminants present.
For Nanodrop, use 1 μL. However, Qubit fluorometer accuracy 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.
?. [DNA PRECIPITATION]
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 extended periods.
?. [PEG small fragment elimination]
Remove the ethanol and allow the pellet to air dry for a couple of minutes. Resuspend pellet in 250 uL 0.1x TE.
?. [PEG small fragment elimination]
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 extended periods.
?. [PEG small fragment elimination]
Combine 250 uL DNA with 250 uL 9% PEG buffer and incubate for 30 min at room temperature.
?. [PEG small fragment elimination]
Centrifuge for 30 min at 10,000 rcf at room temperature.
?. [PEG small fragment elimination]
Carefully remove the supernatant without disturbing the pellet.
?. [PEG small fragment elimination]
Wash pellet by covering it with 300-500 uL 80% Ethanol, incubate for 5 minutes at room temperature then spin for 5 minutes at 10,000 rcf at room temperature. Repeat this step one more time.
?. [PEG small fragment elimination]
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 10-30% recovery relative to total input.For pure DNA, Nanodrop: Qubit quantifications are 1:1.Expect 10-30% recovery relative to total input.