Apr 11, 2024
Version 2
High molecular weight DNA extraction from fungal spores for long read sequencing V.2
- Nonthakorn (Beatrice) Apirajkamol1,2,
- Wee Tek Tay1,2,
- Bishwo Mainali2,
- Phillip Taylor2,
- Thomas Kieran Walsh1,2
- 1CSIRO;
- 2Macquarie University
Protocol Citation: Nonthakorn (Beatrice) Apirajkamol, Wee Tek Tay, Bishwo Mainali, Phillip Taylor, Thomas Kieran Walsh 2024. High molecular weight DNA extraction from fungal spores for long read sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8ybnv5b/v2Version created by Nonthakorn (Beatrice) Apirajkamol
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: April 10, 2024
Last Modified: April 11, 2024
Protocol Integer ID: 98047
Keywords: DNA extraction, PacBio HiFi, Long read sequencing, High molecular weight DNA extraction, Fungi
Abstract
A modified extraction protocol is required to extract high quantity and quality DNA from fungal spores. We optimised DNA extraction protocols to obtain a sufficient amount of high molecular weight DNA from fungal spores for long read sequencing i.e., PacBio HiFi.
Note: If required, the DNA extraction protocol can be scaled up to achieved the desired amount of genomic DNA
Image Attribution
Metarhizium pingshaense (M-1000)
Materials
Samples
Fungal spores
Consumable
Tris-hydrochloride (Tris-HCL)
Ethylenediaminetetraacetic acid (EDTA)
Sodium dodecyl sulfate (SDS)
2-mercaptoethanol (β-mercaptoethanol)
1.0 mm zirconia (ceramic) beads
1.5 and 2ml microcentrifuge tubes
Protease K
RNase A
Sodium acetate
Isopropanol
Ethanol
Auto pipette and pipette tips
Paper towel
TE buffer
Equipment
Tissue homogeniser
Incubator (set for 56-57 and 37˚C)
Vortex
Centrifuge
Heat block (optional)
Cell disruption
Cell disruption
30m
Note: to obtain the best outcome, freshly made lysis buffer should be used.
Make cell lysis buffer: 50mM Tris-HCL pH8.5, 50mM EDTA, 5% SDS, and 1% 2-mercaptoethanol
Add 250 µl of 1.0 mm zirconia (ceramic) beads and 600 µl of cell lysis buffer in a 2ml microcentrifuge tube
Note: 1.0mm zirconia (ceramic) disruptor beads suit for fungal spores size from 2-3.5 μm.
Add spore sample (~50-200 mg)
Homogenise with tissue homogeniser (5,000 rpm for 15 seconds)
15s
To precipitate cell debris, centrifuge at high speed (≥19,000g) for 10-15 minutes or longer if required
15m
Collect supernatants to a new 1.5ml microcentrifuge tube (avoiding cell debris pallet)
RNA and protein removal
RNA and protein removal
3h 30m
Add 20 µl of protease K (20 mg/ml, invitrogen(TM), cat. #25530049) and vortex briefly
Incubate at 56-57℃ for a maximum of 3 hours or until the mixture turns clear
3h
Cool it to 22-24℃ (room temperature)
Add 3 µl of RNase A (100 mg/ml, Qiagen cat. # 19101) and incubate at 37˚C for 5 minutes
Note: If different concentrations of protease K and RNase A were used, the manufacturer’s recommended volume will need to be adjusted accordingly.
5m
To precipitate protein, add half of volume of 3M sodium acetate (pH5.2) to the supernatant
Vortex for 30 seconds (make sure to vortex well, it should get cloudy)
30s
Centrifuge for 5-10 minutes at high speed (≥19,000g) or until the supernatant have no visible cell debris or protein
10m
Transfer supernatant to a new tube (avoiding the precipitated protein pallet)
DNA precipitation
DNA precipitation
1h
To precipitate DNA, add equal volume of isopropanol (≥99.8%) and invert the tube 10x DO NOT VORTEX
Centrifuge for 10-15 minutes at high speed (≥19,000g)
15m
Remove the supernatant using a pipette making sure to avoid disturbing the DNA pallet; invert the tube over a piece of clean absorbant paper to dry the tube and DNA pellet
To wash the DNA pallet, add 1000 µl of freshly made 70% w/v (80% v/v) ethanol (from ≥99.5% undenatured ethanol) and invert the tube gently 10 times DO NOT VORTEX
Centrifuge for 10-15 minutes at high speed (≥19,000g)
15m
Remove the supernatant and dry the tube over paper as described in step# 18
To ensure there is no alcohol residue, dry tubes at room temperature for an hour or in a heat block (56ºC) for no longer than 15 minutes
15m
Add 20-50µl of TE buffer (Invitrogen(TM), cat. #12090015) and leave the DNA pallet to resuspend at room temperature overnight or at 56ºC for 10 minute
Results
Results
Examples of pooled genome of four Metarhizium species. The total amount of DNA extracted per sample ranged between 23-43µg (derived from approximately 500 - 1,000 mg of starting fungal material. 5 times scaled up) and was submitted to Genomics WA (Perth, Australia) for whole genome sequencing. The genomes were sequenced using PacBio HiFi Sequel‱ ll sequencer with SMRTBell technology.
