TITLE: DNA clean-up and size selection for long-read sequencing
AUTHORS: Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>DNA extractions often contain impurities which limit the output of long-read sequencing technologies. Here a protocol is provided which removes impurities and size selects for longer fragments. To remove residual RNA and protein, an additional RNAse A and Proteinase K treatment is performed. A clean-up with chloroform: isoamyl alcohol (24:1) removes these proteins and other hydrophobic organics such as lipids. A low volume ethanol precipitation and wash is used to concentrate the DNA, hopefully also reducing polysaccharides. An optional needle shearing is described which can help create a more uniform DNA length to maximise sequencing output. Finally, polymer and salt based solutions are used to selectively precipitate DNA based on size, which also appears to further clean the DNA. This was trialled for the sorghum rot fungus </span><span style = "font-style:italic;">Macrophomina phaseolina</span><span>, providing highly promising results with an Oxford Nanopore MinION. One strain yielded 13.71 Gbases with an N50 of 21.75 kb, another strain yielded 9.72 Gbases with an N50 of 43.50 kb. Similar results have been obtained with other fungi, plants, reptiles and insects.</span></div></div>

[STEPS]
?. [RNA AND PROTEIN REMOVAL]
Aliquot 10-30 μg of DNA into a 1.5 mL eppendorf tube. Increase volume to 200 μL with 10 mM Tris-HCl pH 8.
DNA quantification must be based on Qubit Fluorometer (Thermo Fisher Scientific), or similar device. A high quantity of RNA may be present. Volume can exceed 200 μL, maximum of 600 μL due to tube capacity at later steps (ethanol precipitation).
?. [RNA AND PROTEIN REMOVAL]
Add RNAse A and Proteinase KEnzymeTarget concentrationStock concentrationQuantity from stockRNAse A100 μg/mL20 mg/mL1 μLProteinase K100 μg/mL20 mg/mL1 μL
EnzymeTarget concentrationStock concentrationQuantity from stockRNAse A100 μg/mL20 mg/mL1 μLProteinase K100 μg/mL20 mg/mL1 μL
?. [RNA AND PROTEIN REMOVAL]
Incubate the samples at 55°C (or 50-60°C) for 20 min, shaking at 400-500 rpm if possible.
?. [CHLOROFORM CLEAN]
Increase the volume to 600 μL with 10 mM Tris-HCl pH 8 (add 400 μL).
?. [CHLOROFORM CLEAN]
Add an equal volume of chloroform: isoamyl alcohol (24:1, v/v) (600 μL) and mix by inverting 10-15 times.
Ensure the organic and aqueous phases become mixed at least temporarily.
?. [CHLOROFORM CLEAN]
Separate the phases by centrifuging at 16,000 rcf for 1 min at 20°C (or room temperature).
?. [CHLOROFORM CLEAN]
Transfer the upper aqueous phase to a new 1.5 mL Eppendorf tube.
?. [CHLOROFORM CLEAN]
Repeat the chloroform: isoamyl alcohol clean (equal volume).
?. [DNA PRECIPITATION]
Add 1.5x volume of 100% ethanol (~900 μL) and 0.1x volume of 3 M sodium acetate pH 5.2 (~60 μL). Mix thoroughly by inverting the tube.
Sample volume should be a maximum 600 μL after cleaning with chloroform: isoamyl alcohol.
?. [DNA PRECIPITATION]
Centrifuge at 16,000 rcf for 1 min at 20°C (or room temperature).
High-molecular weight DNA should pellet easily, therefore you could pulse spin for 10 s at maximum speed. If no pellet can be seen, centrifuge for longer. Bringing ethanol concentration to 2x volume is also an option.
?. [DNA PRECIPITATION]
Add approximately 700 µL of freshly prepared 70% ethanol, enough to cover the pellet.
?. [DNA PRECIPITATION]
Centrifuge at 16,000 rcf for 1 min at 20°C (or room temperature).
?. [DNA PRECIPITATION]
Repeat previous steps for a second 70% ethanol wash.
?. [DNA PRECIPITATION]
Air-dry the pellet by placing the tube upside-down on tissue paper for 5-15 min (until all ethanol has evaporated). Be careful of water touching or dislodging the pellet.
?. [DNA PRECIPITATION]
Dissolve DNA with 60 µL of 10 mM Tris-HCl pH 8.
?. [DNA PRECIPITATION]
Quantify the DNA on a Nanodrop and a Qubit fluorometer (dsDNA broad-range assay).
?. [NEEDLE SHEARING (OPTIONAL)]
Needle shearing is optional and should be skipped in most cases. It can maximise long-read sequencing output at the expense of read length. It is not ideal for genome assembly projects, but it helps provide a more uniform DNA length for specific applications where output is valued more than read length.For instance, repetitive polyploid plant DNA 100 kb and higher can block nanopores, stopping sequencing prematurely. Also, sequencing adapters may not be effectively ligated to these fragments if secondary structures are forming.  Choose a needle and a syringe; increasing needle gauge (smaller diameter), causes more DNA shearing. NeedlePassesEstimated N5029 gauge510-20 kb26 gauge520-35 kb
NeedlePassesEstimated N5029 gauge510-20 kb26 gauge520-35 kb
No difference was found between 5, 10, 15 and 20 passes using the 29 gauge needle with plant DNA. The 26 gauge needle has not been extensivley tested.The chloroform clean-up and DNA precipitation from previous sections has already caused some DNA shearing.
?. [NEEDLE SHEARING (OPTIONAL)]
Aliquot 3-9 μg of DNA into a 1.5 mL Eppendorf DNA LoBind tube. Adjust the volume to exactly 60 μL using 10 mM Tris-HCl (pH 8).
?. [NEEDLE SHEARING (OPTIONAL)]
Connect the needle to a syringe and perform 5 passes with DNA (aspirate and dispense 5 times).
?. [SIZE SELECTION ]
DNA can be size selected by using polymer and salt solutions to preferentially precipitate high-molecular weight DNA, effectively removing shorter fragments. Key solutions are presented here and some commercially available options are also listed. Choose a size selection solution below that suits your experiment, all work on similar principles. These are 2x solutions that will be mixed with an equal volume of DNA at later steps, therefore the final concentrations will be half of what is listed (1x). Solution (2x)Size selectionRecovery4% PVP 360,000, 1.2 M KCl, 20 mM Tris-HCl pH 8> 10 kb50-75%3% PVP 360,000, 1.2 M NaCl, 20 mM Tris-HCl pH 8> 25 kb30-60%9% PEG 8,000, 1 M NaCl, 10 mM Tris-HCl pH 8>10 kbNeeds more testingShort-Read Eliminator XS (Circulomics)> 10 kb50-75%Short-Read Eliminator (Circulomics)> 25 kb30-60%Short-Read Eliminator XL (Circulomics)> 40 kb
Solution (2x)Size selectionRecovery4% PVP 360,000, 1.2 M KCl, 20 mM Tris-HCl pH 8> 10 kb50-75%3% PVP 360,000, 1.2 M NaCl, 20 mM Tris-HCl pH 8> 25 kb30-60%9% PEG 8,000, 1 M NaCl, 10 mM Tris-HCl pH 8>10 kbNeeds more testingShort-Read Eliminator XS (Circulomics)> 10 kb50-75%Short-Read Eliminator (Circulomics)> 25 kb30-60%Short-Read Eliminator XL (Circulomics)> 40 kb
Size selection and recovery estimates are based on high-molecular weight DNA from non-model species; results may vary considerably. Expect higher loss if DNA is significantly sheared. If the DNA sample is very contaminated, size selection may perform poorly or the DNA may not precipitate.
?. [SIZE SELECTION ]
Aliquot 3-9 μg of DNA into a 1.5 mL Eppendorf DNA LoBind tube. Adjust the volume to exactly 60 μL using 10 mM Tris-HCl pH 8 (alternatively buffer EB if using a Circulomics product).
DNA mass must be measured by Qubit Fluorometer (Thermo Fisher Scientific) or equivalent.DNA must not contain high levels of salts, polyphenols, polysaccharides or other contaminants.If you want to add more DNA, increase the volume slightly (also more size selection solution later). For example, 12 μg of DNA in 100 μL (adding 100 μL of size selection solution later).DNA must not contain high levels of salts, polyphenols, polysaccharides or other contaminants.If you want to add more DNA, increase the volume slightly (also more size selection solution later). For example, 12 μg of DNA in 100 μL (adding 100 μL of size selection solution later).
?. [SIZE SELECTION ]
Add 60 μL (an equal volume) of the chosen size selection solution. Mix thoroughly by gently tapping the tube or by gently pipetting up and down.
?. [SIZE SELECTION ]
Centrifuge at 10,000 rcf for 30 min at 20°C (or room temperature). Note where the DNA will pellet, by marking the tube or placing the tubes in a consistent orientation within the rotor.
Recovery will be impacted if centrifugation is performed at low temperature (e.g. 4°C).
?. [SIZE SELECTION ]
Carefully remove the supernatant with a pipette as soon as possible, without disturbing the pellet. Care must be taken as the pellet is fragile, being easily dislodged.
The DNA pellet may not be visible. Be careful of accidentally losing the pellet; mark the expected spot and proceed as if it was there.It is good practice to save the supernatent in another tube, in case the DNA has not precipitated or the pellet has been unknowingly dislodged.
?. [SIZE SELECTION ]
Add 200 μL of freshly prepared 70% ethanol, enough to cover the pellet. Do not tap or mix after adding ethanol.
?. [SIZE SELECTION ]
Centrifuge at 10,000 rcf for 2 min at at 20°C (or room temperature).
?. [SIZE SELECTION ]
Repeat previous steps for a second 70% ethanol wash.
?. [SIZE SELECTION ]
Air-dry the pellet by placing the tube upside-down on tissue paper for 5-15 min at room temperature (until all ethanol has evaporated). Be careful of water touching or dislodging the pellet.
?. [SIZE SELECTION ]
Let the pellet hydrate at room temperature, may take 10 min. If it does not hydrate, incubate at 50°C for 10 min.
Circulomics protocols recommend 50°C for 1 h, which seems excessive. DNA concentration was found to increase
?. [SIZE SELECTION ]
After incubation, gently tap the tube to ensure that the DNA is properly resuspended and mixed.
?. [SIZE SELECTION ]
Quantify the DNA on a Nanodrop and a Qubit fluorometer (dsDNA broad-range assay).
?. [SIZE SELECTION ]
Store DNA at 4°C (in the fridge) 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.
?. EXPECTED RESULTSFresh mycelia (approximately 2 g) was used to extract crude high-molecular weight DNA using a CTAB method previously described. See Jones et al. (2019) High-molecular weight DNA extraction from challenging fungi using CTAB and gel purification, Protocols.io.The DNA was then cleaned and size selected by following the presented protocol in full, with the final size selection being performed with a Short-Read Eliminator (SRE) (Circulomics, > 25 kb selection). Initially the Qubit:Nanodrop ratio had a large discrepancy (Table 1 and Figure 1). This is due to the presence of RNA, given the 260/280 is above 1.8. Cleaning removed this discrepancy, particularly with the removal of all RNA. With RNA not present, the 260/230 ratio is more representative, despite appearing worse. For sequencing, we adopted a MinION sequencer from Oxford Nanopore Technologies, creating a native genomic DNA library (SQK-LSK109) following the manufacturer’s instructions. Although the DNA was not perfect, a high sequencing output was achieved (Table 2). Size selection with SRE resulted in long-reads and a high N50 (Figure 2).
?. [DNA PRECIPITATION]
Incubate the sample on ice for 1 min.
?. [DNA PRECIPITATION]
Carefully decant the supernatant as soon as possible, without disturbing the pellet.
?. [NEEDLE SHEARING (OPTIONAL)]
Proceed directly to size selection.
?. [SIZE SELECTION ]
Carefully remove the supernatant with a pipette as soon as possible, without disturbing the pellet.
?. [DNA PRECIPITATION]
Carefully decant the supernatant as soon as possible, without disturbing the pellet.
?. [DNA PRECIPITATION]
Let the pellet soak for 1 min at room temperature to dissolve excess salts.
?. [SIZE SELECTION ]
Add 50 μL of 10 mM Tris-HCl pH 8 (alternatively buffer EB if using a Circulomics product). Mix by gently tapping the tube.