TITLE: High molecular weight DNA extraction from fungal spores for long read sequencing
AUTHORS: Nonthakorn (Beatrice) Apirajkamol, Wee Tek Tay, Bishwo Mainali, Phillip Taylor, Thomas Kieran Walsh

[DESCRIPTION]
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

[STEPS]
SECTION: Cell disruption
1. 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 
SECTION: Cell disruption
2. 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. 
SECTION: Cell disruption
3. Add spore sample (~50-200 mg) 
SECTION: Cell disruption
4. Homogenise with tissue homogeniser (5,000 rpm for 15 seconds) 
SECTION: Cell disruption
5. To precipitate cell debris, centrifuge at high speed (≥19,000g) for 10-15 minutes or longer if required
SECTION: RNA and protein removal
7. Add 20 µl of protease K (20 mg/ml, invitrogen(TM), cat. #25530049) and vortex briefly
SECTION: Cell disruption
6. Collect supernatants to a new 1.5ml microcentrifuge tube (avoiding cell debris pallet) 
SECTION: RNA and protein removal
8. Incubate at 56-57℃ for a maximum of 3 hours or until the mixture turns clear
SECTION: RNA and protein removal
10. 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. 
SECTION: RNA and protein removal
9. Cool it to 22-24℃ (room temperature)
SECTION: RNA and protein removal
11. To precipitate protein, add half of volume of 3M sodium acetate (pH5.2) to the supernatant 
SECTION: RNA and protein removal
12. Vortex for 30 seconds (make sure to vortex well, it should get cloudy)
SECTION: RNA and protein removal
13. Centrifuge for 5-10 minutes at high speed (≥19,000g) or until the supernatant have no visible cell debris or protein
SECTION: RNA and protein removal
14. Transfer supernatant to a new tube (avoiding the precipitated protein pallet)
SECTION: DNA precipitation
15. To precipitate DNA, add equal volume of isopropanol (≥99.8%) and invert the tube 10x DO NOT VORTEX
SECTION: DNA precipitation
22. 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

SECTION: DNA precipitation
21. 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 
SECTION: DNA precipitation
20. Remove the supernatant and dry the tube over paper as described in step# 18
SECTION: DNA precipitation
19. Centrifuge for 10-15 minutes at high speed (≥19,000g)
SECTION: DNA precipitation
18. 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
SECTION: DNA precipitation
17. 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
SECTION: DNA precipitation
16. Centrifuge for 10-15 minutes at high speed (≥19,000g)
SECTION: Results
23. 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.