High molecular weight DNA extraction from fungal spores for long read sequencing
Nonthakorn (Beatrice) Apirajkamol, Wee Tek Tay, Bishwo Mainali, Phillip Taylor, Thomas Kieran Walsh
Disclaimer
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
Before start
Steps
Cell disruption
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% beta- 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)
To precipitate cell debris, centrifuge at high speed for 10-15 minutes or longer if required
Collect supernatants to a new 1.5ml microcentrifuge tube (avoiding cell debris pallet)
RNA and protein removal
Add 20 µl of protease K (20 mg/ml, invitrogenTM, cat. #25530049) and vortex briefly
Incubate at 56-57℃ for a maximum of 3 hours or until the mixture turns clear
Cool it at room temperature
Add 3 µl of RNase (100 mg/ml, Qiagen cat. # 19101) and incubate at 37˚C for 5 minutes
Note: If different concentrations of protease K and RNase were used, the manufacturer’s recommended volume will need to be adjusted accordingly.
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)
Centrifuge for 5-10 minutes at high speed or until the supernatant have no visible cell debris or protein
Transfer supernatant to new tube (avoiding precipitated protein pallet)
DNA precipitation
To precipitate DNA, add equal amounts of isopropanol and invert the tube 10x DO NOT VORTEX
Centrifuge for 10-15 minutes at high speed
Remove supernatant and dry tube over paper (please use pipette to avoid disturbing DNA pallet)
To wash DNA pallet, add 1000 µl of freshly made 70% (80% v/v) ethanol and invert the tube 10x DO NOT VORTEX
Centrifuge for 10-15 minutes at high speed
Remove supernatant and dry tube over paper (again, please use pipette to avoid disturbing DNA pallet)
To ensure there is no alcohol residue, dry tubes at room temperature for an hour or in heat block (56ºC) for no longer than 15 minutes
Add 20-50µl of TE buffer (InvitrogenTM, cat. #12090015) and leave the DNA pallet to dissolve at room temperature overnight or at 56ºC for no longer than 10 minute
Results
Example of four Metarhizium spp. genomes. 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.
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#尊敬的用户，由于网络监管政策的限制，部分内容暂时无法在本网站直接浏览。我们已经为您准备了相关原始数据和链接，感谢您的理解与支持。
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