TITLE: HMW DNA extraction from diverse plants species for PacBio and Nanopore sequencing
AUTHORS: Alessia Russo, Giacomo Potente, Baptiste Mayjonade

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Modified from the protocol of Baptiste Mayjonade, Jérome Gouzy, Cécile Donnadieu, Nicolas Pouilly, William Marande, Caroline Callot, Nicolas Langlade and Stéphane Munos, High molecular weight gDNA extraction, Bio Techniques, Vol. 61, No. 4, October 2016, pp. 203-205. BioTechniques 61:203-205 (October 2016) doi 10.2144/000114460</div><div class = "text-block"><a href="https://www.future-science.com/doi/10.2144/000114460" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.future-science.com/doi/10.2144/000114460</span></a></div><div class = "text-block">The original protocol failed to extract High Molecular Weight genomic DNA from orchids samples (poor yield and low purity). Therefore modifications (mainly/mostly BME addition during the lysis step and a Phenol:Chloroform purification step) were made to create a new version of this protocol. This new version allowed to successfully extract DNA from orchid samples. This DNA was then sequenced on a Nanopore PromethION platform and good results in term of total yield and read length were obtained. This protocol was also successfully applied to a wide range of plant species (See attached document/excel sheet).</div><div class = "text-block"><span style = "font-weight:bold;">ACKNOWLEDGEMENT</span></div><div class = "text-block"><span>Sequencing of the </span><span style = "font-style:italic;">Arabidopsis thaliana</span><span> samples listed in the attached reports was performed in collaboration with the GeT core facility, Toulouse, France (</span><a href="http://get.genotoul.fr)" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://get.genotoul.fr)</span></a><span>,  and was supported by France Génomique National infrastructure, funded  as part of “Investissement d’avenir” program managed by Agence Nationale  pour la Recherche (contract ANR-10-INBS-09). We particularly want to thank Lisa Gil and Céline Lopez-Roques for the sequencing part and Roxane Boyer for the data processing.</span></div><div class = "text-block"><span>Sampling and DNA Extraction for the </span><span style = "font-style:italic;">Ophrys sphegodes </span><span>samples were financially supported by the University of Zürich Research Priority Programme URPP "Evolution in Action" (PMS/AR). Sequencing was performed by Daniel Frei in the Department of Molecular Diagnostics, Genomics and Bioinformatics, Agroscope Wädenswil, Switzerland. We are thankful to Jürg E. Frey, Agroscope, for financial support. We are grateful to Philipp M. Schlüter and Ueli Grossniklaus for support and helpful discussion.</span></div><div class = "text-block"><span>Sequencing of </span><span style = "font-style:italic;">Lolium multiflorium </span><span>was performed in Agroscope by Daniel Frei. We thank Daniel, Jürg E. Frei, Dario Copetti and Bruno Studer, Department of Environmental Systems Science,  ETH Zürich, for sharing the sequencing results.</span></div><div class = "text-block"><span>Sequencing of </span><span style = "font-style:italic;">Brassica incana </span><span>was performed at FGCZ Zürich. We are thankful to Léa Frachon, Department of Systematic and Evolutionary Botany, UZH Zürich, to share the data.</span></div><div class = "text-block"><span>DNA extraction of </span><span style = "font-style:italic;">Gorteria diffusa </span><span>was performed by Roman T. Kellenberger, Department of Plant Sciences, University of Cambridge. We wish to thank him for sharing his sequencing results. </span></div></div>

[STEPS]
?. [Cell Lysis]
Incubate Lysis Buffer at 65 °C for at least 30 minutes (gently mix the tube every 10 minutes). For convenience, one can aliquote 600 µl Lysis Buffer in a 2 ml tube per sample and incubate in the water bath.Keep the SDS Lysis Buffer at 65°C until the tissue powder is added (the buffer has to be warm to ensure that powder is well dissolved).
Work under a fume hood until the end of the protocol because of the presence of BME, Phenol and Chloroform.
?. [Cell Lysis]
Carefully add ~ 50-100 mg tissue powder (1-2 spoons SP SCIENCEWARE 0.10 ml, see equipment list) to a 2 ml tube with 600 µl of warm SDS Lysis Buffer.Immediately homogenize by vortexing 3-5 seconds. It’s important to rapidly homogenize the lysate to neutralize DNase, polyphenols,...
Add more than 100 mg of tissue can decrease DNA purity (A260/230).
?. [Cell Lysis]
Incubate 20 min @ 55°C with gentle agitation in a thermomixer (maximum 400 rpm) or in a water bath. Invert the tube gently 10 times after 10 min.
?. [Cell Lysis]
Add 4 µl 100mg/ml RNase A, gently mix by inversion 10 times and incubate 10 minutes at 55 °C with gentle agitation.
?. [Contaminant precipitation and phenol:chloroform purification]
Add 200 µl 5M KAc. Mix by gently pipetting up and down 5 times with a P1000 cut tip and by inverting the tube 5 times in order to obtain a homogeneous solution to fully precipitate the proteins and the polysaccharides that will complex with SDS.
?. [Contaminant precipitation and phenol:chloroform purification]
Phenol:Chloroform purification.Option 1: Add 800 µl Phenol:Chloroform:IsyamylAlcohol pH=8.Option 2: If you don't have a ready-to-use Phenol:Chloroform:IsyamylAlcohol solution, add 400 µl Phenol pH=8, mix by inverting and add 400 µl Chloroform:IsoamylAlcohol 24:1.
Work under a fume hood.
?. [Contaminant precipitation and phenol:chloroform purification]
Incubate 10 min @ 20rpm/min on a rotator.
?. [Contaminant precipitation and phenol:chloroform purification]
Centrifuge 10 minutes @ 10,000g at RT.
?. [Contaminant precipitation and phenol:chloroform purification]
Transfer the supernatant (usually 700-800 µl) in a new 2 ml tube with a P1000 cut tip. Pipette slowly and with a cut tip to avoid breaking the long DNA fragments. Make sure not to carry over the interphase or the lower layer that would contaminate the DNA.
?. [Contaminant precipitation and phenol:chloroform purification]
Add the same volume of Chloroform:IsoamylAlcohol 24:1
?. [Contaminant precipitation and phenol:chloroform purification]
Incubate at RT for 10 min with gentle agitation (20 rpm)
?. [Contaminant precipitation and phenol:chloroform purification]
Centrifuge RT 10,000g for 10 min
?. [Contaminant precipitation and phenol:chloroform purification]
Transfer the supernatant into a new 2 ml tube. Make sure not to carry over the interphase or lower layer that would contaminate the DNA.Determine the final volume.
?. [Genomic DNA Purification]
Before starting, vortex the Bead Solution for 30 seconds to ensure beads are completely resuspended.Add 1 volume of Bead Solution to the sample tube (1:1 ratio, v/v).
?. [Genomic DNA Purification]
Incubate @10 rpm on a rotator for 20-30 min at RT.
?. [Genomic DNA Purification]
Spin down for 1 second.
?. [Genomic DNA Purification]
Place the tube on a magnetic rack and wait until all beads bind at the back of the tube and the solution becomes clear (this will probably take a few minutes, as the DNA is highly concentrated and HMW).
?. [Genomic DNA Purification]
Remove the supernatant without disturbing the bead pellet.
?. [Genomic DNA Purification]
Add 1 ml EtOH 70% (the washing solution).
?. [Genomic DNA Purification]
Remove the tube from the magnetic rack and mix by gently inverting the tube 20 times to resuspend the bead pellet in the washing solution (probably it won't be entirely resuspendend and there will be some small/big bead aggregates floating - if that happens, don't worry).
?. [Genomic DNA Purification]
Spin down for 1 second.
?. [Genomic DNA Purification]
Place the tube on a magnetic rack and wait until all beads bind at the back of the tube and the solution becomes clear.
?. [Genomic DNA Purification]
Remove the supernatant without disturbing the bead pellet.
?. [Genomic DNA Purification]
Repeat steps 20 - 24 two-three times (total possible washing = 4 times, but usually 2 washes are enough).
?. [Genomic DNA Purification]
Spin down for 1 second and remove the remaining washing solution with a P10 pipette.
?. [Genomic DNA Purification]
Add 50 µl EB (or Tris-HCl 10 mM pH=8.5).
?. [Genomic DNA Purification]
Resuspend the beads by gently swirling or flicking the tube. This time they must be completely resuspended. It is important that the beads are not aggregated, or the elution efficiency will be reduced.
?. [Genomic DNA Purification]
Incubate at 37 °C for 15 minutes and flick the tube again.
?. [Genomic DNA Purification]
Spin down for 1 second.
?. [Genomic DNA Purification]
Place the tube on a magnetic rack and wait until all beads bind at the back of the tube and the solution becomes clear. If DNA is at high concentration, this can take a long time. You can leave it overnight at RT, and/or add more EB (if you do so mix by flicking the tube after the addition of EB and put the tube back on the Magnetic Rack).
?. [Genomic DNA Purification]
Transfer the eluted DNA into a new tube, with a cut tip, or using a wide-bore tip, as DNA is very concentrated and the solution very viscous.
?. [Genomic DNA Purification]
Leave the DNA at RT overnight, as the HMW DNA is very aggregated and needs to get relaxed and homogenized. The day after proceed with Nanodrop lecture, to determine the purity, and Qubit to determine concentration.
?. [Cell Lysis]
Grind ~100-110 mg leaf tissue in LN2 with mortar and pestle. This is a crucial step as it will affect final DNA yield, purity, and integrity.Plant leaves must be flash frozen in LN2 immediately after harvesting and stored at -80 °C until DNA extraction procedure, if not immediately used. Here a useful tutorial on how to grind plant tissue with LN2 https://bionanogenomics.com/videos/liquid-nitrogen-grinding-tutorial/ . As they say: "Keep grinding until powder is as fine as wheat flour"Tips to achieve better results: - Apply enough pressure to allow cells to break and recover as much DNA as possible. - Keep the powder frozen until it goes into the lysis buffer, by adding LN2 while grinding (LN2 evaporates quickly). This prevents nuclease activation and oxidation processes (once powder is in the lysis buffer, the nucleases will be inactivated by the SDS and EDTA, and the oxidation blocked by BME and Metabisulfite)
?. [Additional Purification]
An additional purification steps can improve the A260/A280 and A260/A230 ratios. At this step you can pool the elution of multiple tubes.Before starting: - Incubate the Beads Solution at RT for more than 30 minutes prior to use;- Mix the beads by vortexing for 20 seconds before use .Repeat steps 15 to 33.
?. [QC]
Determine the purity of the DNA at 230, 260, 280 nm with a spectrophotometer (e.g. NanoDrop 2000). Acceptable values are:- A260/280 = 1.8 - 2.0;- A260/230 = 2.0 - 2.2.A260/280 >2.0 or A260/230 >2.2 could be indicative of a contamination by RNA.
?. [QC]
Determine the concentration of double-stranded DNA with a fluorimetric-based method (e.g. Qubit 3.0 Fluorometer BR assay). The ratio between the concentration measured with Nanodrop, over the concentration measured by Qubit should be
?. [QC]
Evaluate DNA integrity on a TapeStation, and fragment length on PhemtoPulse (or PFGE).