Jun 08, 2020

Public workspaceHigh-molecular weight DNA extraction and small fragment removal of Ascochyta lentis

 Forked from High-molecular weight DNA extraction from challenging fungi using CTAB and gel purification
DOI
dx.doi.org/10.17504/protocols.io.bgw5jxg6
  • 1Curtin University;
  • 2Australian National University;
  • 3ICAR-National Institute for Plant Biotechnology
Johannes Wolfram JWD Debler
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bgw5jxg6
Protocol Citation: Johannes Wolfram JWD Debler, Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger 2020. High-molecular weight DNA extraction and small fragment removal of Ascochyta lentis. protocols.io https://dx.doi.org/10.17504/protocols.io.bgw5jxg6
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: May 28, 2020
Last Modified: June 16, 2020
Protocol Integer ID: 37565
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 small fragment removal step using Polyethylene Glycol. Using this protocol, we have been successfully sequencing the lentil pathogen Ascochyta lentis 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.

DNA extraction modified from: https://www.protocols.io/view/high-molecular-weight-dna-extraction-from-challeng-5isg4ee

PEG small fragment elimination after: https://www.protocols.io/view/size-selective-precipitation-of-dna-using-peg-amp-7erhjd6
Guidelines
This research builds on the work of Ramawater Nagar and Benjamin Schwessinger, as well as John Tyson; 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.

https://www.longreadclub.org/mountain-protocol/
Materials

Reagents
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
Polyethylene Glycol 8000
Yeast Extract
Glucose

Special Equipment
1.5 mL DNA LoBind Eppendorf tube (optional)
Centrifuge for 15 mL Falcon tubes (up to 5,000 rcf)
Mortar and pestle
Water bath
PREPARATION
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.
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 500 mg of sample.

ComponentMWStockQuantity (5 mL)
2% CTAB (w/v)364.45powder0.10 g
100 mM Tris-HCl (pH 8)157.601 M0.50 mL
20 mM EDTA (pH 8)292.240.5 M0.20 mL
1.2 M NaCl58.445 M1.20 mL
1% PVP40,00010%0.50 mL
Milli-Q water--2.60 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 500 mg of sample.
ComponentMWStockQuantity (10 mL)
2% CTAB (w/v)364.45powder0.20 g
100 mM Tris-HCl (pH 8)157.601 M1 mL
20 mM EDTA (pH 8)292.240.5 M0.40 mL
Milli-Q water--8.60 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.

9% PEG8000 small fragment elimination buffer
  • ~300 uL per 500 mg of sample.


ComponentMWStockQuantity (10 mL)
9% PEG 8000800018%5 mL
1M NaCl58.445 M2 mL
10 mM Tris-Cl (pH 8.0)157.601 M100 uL
Milli-Q water--2.9 mL

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.
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.

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.

Transfer the ground mycelium to 5 mL lysis buffer in a 15 mL falcon tube.

Add RNAse A and Proteinase K to the solution:

EnzymeStockQuantity (5 mL lysis buffer)
100 μg/mL RNAse A10 mg/mL50 μL
100 μg/mL Proteinase K20 mg/mL25 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
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.


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.
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 5,000 rcf for 30 minutes at 20°C to pellet crystals.


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 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.
 Centrifuge at 5,000 rcf for 10 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 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.
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.

  • This step can take a couple of days!

Transfer DNA to a 1.5 mL DNA LoBind Eppendorf tube.
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.

PEG small fragment elimination
PEG small fragment elimination
Combine 250 uL DNA with 250 uL 9% PEG buffer and incubate for 30 min at room temperature.
Centrifuge for 30 min at 10,000 rcf at room temperature.
Carefully remove the supernatant without disturbing the pellet.
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.
Remove the ethanol and allow the pellet to air dry for a couple of minutes. Resuspend pellet in 250 uL 0.1x TE.
Note
  • It can take a couple of days for the pellet to fully rehydrate.

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.