Jul 26, 2019
Version 2
High-molecular weight DNA extraction from challenging fungi using CTAB and gel purification V.2
- 1Australian National University;
- 2ICAR-National Institute for Plant Biotechnology
- High molecular weight DNA extraction from all kingdomsTech. support email: See@each.protocol
External link: https://doi.org/10.1186/s13059-022-02658-2
Protocol Citation: Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger 2019. High-molecular weight DNA extraction from challenging fungi using CTAB and gel purification. protocols.io https://dx.doi.org/10.17504/protocols.io.5isg4ee
Manuscript citation:
Duan H, Jones AW, Hewitt T, Mackenzie A, Hu Y, Sharp A, Lewis D, Mago R, Upadhyaya NM, Rathjen JP, Stone EA, Schwessinger B, Figueroa M, Dodds PN, Periyannan S, Sperschneider J, Physical separation of haplotypes in dikaryons allows benchmarking of phasing accuracy in Nanopore and HiFi assemblies with Hi-C data. Genome Biology doi: 10.1186/s13059-022-02658-2
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 15, 2019
Last Modified: July 26, 2019
Protocol Integer ID: 25906
Abstract
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 gel purification with a Pippin Prep (Sage Science). Using this protocol, we have been successfully sequencing the wheat stripe rust Puccinia striiformis and leaf rust Puccinia triticina with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 4 gigabases with an N50 of approximately 30 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants.
Guidelines
This research builds on the work of Ramawater Nagar and Benjamin Schwessinger; a warm thanks for their contributions.
This protocol is based on the following protocols below.
Nagar, R. and Schwessinger, B. (2018). Multi-step high purity high molecular weight DNA extraction protocol from challenging fungal tissues. Protocols.io
Arseneau et al. (2017). Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues. Molecular Ecology Resources 17(4), 686-693.
Xin, Z. and Chen, J. (2012). A high throughput DNA extraction method with high yield and quality. Plant Methods 8, 26.
Materials
Reagents
AMPure XP beads (Agencourt) (or equivalent)
Cetrimonium bromide (CTAB)
Chloroform: isoamyl alcohol (24:1)
Ethylenediaminetetraacetic acid (EDTA)
Ethanol (70%)
Sodium chloride (NaCl)
Proteinase K (20 mg/mL)
Polyvinylpyrrolidone (PVP) 40,000
RNAse A (20 mg/mL)
Trisaminomethane hydrochloride (Tris-HCl) pH 8
Water, high-purity (e.g. Milli-Q system)
Water, nuclease-free
Special Equipment
1.5 mL DNA LoBind Eppendorf tube (optional)
Acid-washed autoclaved sand (fine)
Centrifuge for 50 mL Falcon tubes (up to 16,000 rcf)
Magnetic rack for 1.5 mL Eppendorf tubes
Mortar and pestle
PippinHT (Sage Science) or equivalent gel-purification system
PippinHT 0.75% agarose casette and 15-20 kb kit (Sage Science)
Water bath
PREPARATION
PREPARATION
Prepare up to 600 mg of fungal spores, taking into consideration the genome size (larger genome would need less spores). Keep frozen in liquid nitrogen.
Set a water bath to 55°C. This will be used to dissolve 2% CTAB solutions and to preciptate DNA with CTAB.
Freshly prepare lysis and precipitation buffers.
Lysis buffer
- 5 mL per 100 mg of sample.
- 600 mg of sample is recommended for challenging fungi with a small genome (~50 MB).
- Therefore a total 30 mL of lysis buffer is required.
| Component | MW | Stock | Quantity (5 mL) | Quantity (30 mL) | |
| 2% CTAB (w/v) | 364.45 | powder | 0.10 g | 0.60 g | |
| 100 mM Tris-HCl (pH 8) | 157.60 | 1 M | 0.50 mL | 3 mL | |
| 20 mM EDTA (pH 8) | 292.24 | 0.5 M | 0.20 mL | 1.20 mL | |
| 1.2 M NaCl | 58.44 | 5 M | 1.20 mL | 7.20 mL | |
| 1% PVP | 40,000 | 10% | 0.50 mL | 3 mL | |
| Milli-Q water | - | - | 2.60 mL | 15.60 mL |
Note
- EDTA is a metal ion chelator. By binding to Mg2+, DNase activity is stopped as it is dependent on Mg2+.
Precipitation buffer
- 10 mL per 100 mg of sample.
- 60 mL is recommended to process 600 mg of sample.
- Prepare 30 mL twice in two separate 50 mL Falcon tubes.
| Component | MW | Stock | Quantity (10 mL) | Quantity (30 mL) (prepare twice) | |
| 2% CTAB (w/v) | 364.45 | powder | 0.20 g | 0.60 g | |
| 100 mM Tris-HCl (pH 8) | 157.60 | 1 M | 1 mL | 3 mL | |
| 20 mM EDTA (pH 8) | 292.24 | 0.5 M | 0.40 mL | 1.20 mL | |
| Milli-Q water | - | - | 8.60 mL | 25.80 mL |
Note
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.
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
SAMPLE LYSIS
Place a sterile mortar and pestle into an insulated container. Pour liquid nitrogen into the container to chill the mortar and pestle. Add 1-2 g of acid-washed autoclaved sand to the mortar.
Note
- The lid of an ice box can be used. If liquid nitrogen is later poured directly onto the spores, spores disperse all over the pestle and potentially the table.
- For less than 500 mg of spores, add 1 g of sand . For 500 mg or more spores, use 2 g of sand.
Add spores to the mortar and grind for approximately 1-2 min. Keep the mortar and pestle chilled by having liquid nitrogen within the surrounding container.
Transfer the ground spores and sand to the lysis buffer.
Add RNAse A and Proteinase K to the solution:
| Enzyme | Stock | Quantity (5 mL lysis buffer) | Quantity (30 mL lysis buffer) | |
| 100 μg/mL RNAse A | 20 mg/mL | 25 μL | 150 μL | |
| 100 μg/mL Proteinase K | 20 mg/mL | 25 μL | 150 μL |
Note
- 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).
Vortex and invert the solution. Incubate at 55°C for at least 1 h.
CHLOROFORM CLEAN-UP
CHLOROFORM CLEAN-UP
If the sample occupies more than 50% of the tube capacity, split the sample evenly across two 50 mL Falcon tubes (or appropriate sized tube).
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.
Note
Approximately 5 mL per 100 mg of sample processed.
Separate the phases by centrifuging at 5,000 rcf for 10 min at 20°C. Transfer the upper aqueous phase to a new 50 mL Falcon tube.
Note
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.
Repeat the chloroform: isoamyl alcohol clean (equal volume).
DNA PRECIPITATION
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.
Centrifuge at 16,000 rcf for 10 minutes at 20°C to pellet crystals.
Note
Could spin at 5,000 rcf for 5 min to pellet only high-molecular weight DNA.
Carefully decant the supernatant as soon as possible, without disturbing the pellet. Care must be taken as the pellet is fragile, being easily dislodged.
Add approximately 10-15 mL freshly prepared 70% ethanol, enough to cover the pellet. Let the pellet soak for 15 min at room temperature to dissolve excess salts and CTAB.
Centrifuge at 16,000 rcf for 5 min. Carefully decant the supernatant as soon as possible, without disturbing the pellet.
Air-dry the pellet for 10-15 min, or until all ethanol has evaporated.
Dissolve DNA with a maximum of 200 µL nuclease-free water. If the sample was previously split across two 50 mL Falcon tubes, add 100 µL to each. 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.
Note
- 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.
Transfer DNA to a 1.5 mL DNA LoBind Eppendorf tube. Combine any samples that were previously split.
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.
Note
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.
Store DNA at 4°C to prevent cycles of freeze-thawing that shear the DNA.
Note
No effects on DNA integrity have been noticed for samples stored at 4°C for extended periods.
GEL PURIFICATION: PIPPIN PREP
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.
After separation, wait at least 45 min (hours or overnight is suitable), to aid elution and recovery.
Collect the contents of all elution wells into a 1.5 mL DNA LoBind Eppendorf tube (approx. 300 µL).
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).
Add 1.2x volume of AMPure XP beads (approx. 720 µL). Incubate at room temperature for 5 min.
Note
Alternatively, add 1.2x binding buffer (approx. 720 µL), and 100 µL of 2% Sera-Mag beads.
Place on a magnetic rack for 5 min, or until the solution becomes clear. Discard the supernatant.
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.
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.
Remove the tube from the magnetic rack and elute with 50 µL of nuclease-free water. Incubate for 10 min at room temperature.
Note
A maximum DNA volume of 48 µL is used in an Oxford Nanopore ligation prep (e.g. SQK-LSK109).
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.
Add an additional 50 µL of nuclease-free water to the beads, remove from magnet, resuspend, incubate, magnetise and transfer to a new 1.5 mL Eppendorf tube.
Note
Save this second elution for other applications such as running a pulse field gel.
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.
Note
- 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.
Store DNA at 4°C to prevent cycles of freeze-thawing that shear the DNA.
Note
No effects on DNA integrity have been noticed for samples stored at 4°C for extended periods.
SEQUENCING LIBRARY PREPARATION
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:
| Fragment sizes | Rapid SQK-RAD004 | Ligation SQK-LSK109 | |
| Amplicon or digest (~1 kb) | Not recommended | 200 ng | |
| No size selection (~8 kb) | 800 ng | 2,000 ng | |
| 15 kb gel purification | 1,000 ng | 3,000 ng | |
| 20 kb gel purification | 1,200 ng | 3,000 ng | |
| 30 kb gel purification | Not recommended | 4,000 ng | |
| 40 kb gel purification | Not recommended | 5,000 ng |
Load the MinION and perform sequencing according to Oxford Nanopore's instructions. Ensure no air bubbles are introduced into the array during loading.
Note
Air bubbles destroy nanopores, significantly reducing sequencing yield.
EXPECTED RESULTS
Using the protocol described, we have been obtaining clean high-molecular weight DNA (Table 1, Figure 1). DNA fragment size ranges are above 10 kb in length (Figure 2). During sequencing, we can approximately obtain over 4 gigabases of sequencing from a single MinION revD flow cell (Table 2, Figure 3). This includes quality reads over 100 kb in length and N50 values of approximately 30 kb. An elusive contaminant is present amongst the DNA, which is affecting sequencing yield. More work is needed to identify and remove the contaminant, to increase sequencing yield. A troubleshooting guide for MinION sequencing is presented in Figure 4, illustrating the most common problems researchers have.