TITLE: Bacterial and fungal DNA extraction protocol for long-read whole genome sequencing
AUTHORS: Manon Norest, Anne-Yvonne Guillerm-Erckelboudt, Claudia Bartoli 

[DESCRIPTION]
In the context of the ERC Starting Grant HoloE2Plant N° 101039541 “Exploring the Holobiont concept through a Plant Evolutionary Experiment study”, long read bacterial and fungal reference genomes were performed with PacBio Sequel II technology. The purpose of these genomes is to help in the Synthetic Microbial Communities (SynComs) construction. As DNA extraction is a crucial step prior to obtain high-quality genomes, we developed a two-days DNA extraction protocol efficient for the extraction of High-Molecular-Weight (HMW) genomic DNA on both gram-positive/negative bacteria and fungi. To our knowledge, this is the first protocol enabling to extract HMW DNA from both bacteria and fungi. Our protocol was adapted from the four protocols provided in the references.

[STEPS]
SECTION: Bacterial and fungal growth
2. Fungal growth

Place a fungal stab (obtained from the culture collection tube stored at 4°C) on four Inhibitory Mold Agar plates. Collect the mycelium from the four plates into a 5ml Eppendorf tube and incubate it at -80°C for 24h. Lyophilize the mycelium for 48h at -55°C. In this study we utilized the Heto PowerDry LL3000 freeze
dryer of ThermoScientific.

SECTION: Bacterial and fungal growth
1. Bacterial growth

Bacteria growth can be realized on Petri dishes (solid medium) or in a liquid medium (into 15ml tubes). Both techniques give high quality HMW bacterial DNA. 

To grow a bacterium into Petri dishes, streak the bacterial strain from the collection tube (normally placed at -80°C) onto Nutrient Agar plates with the addition of CaCl2. Incubate the bacteria at the appropriate
temperature.

To grow a bacterium in liquid conditions, inoculate a single bacterial colony (previously obtained from a Nutrient Agar plate) into 10 mL of Nutrient Broth. Incubate the bacterial strain at appropriate temperature under constant agitation (120 rpm) until the stationary phase is reached.
SECTION: Bacterial and fungal lysis
5. Fungal lysis

Weigh 30 mg of dried mycelium and place it into the 2ml Eppendorf tube containing the glass beads. Add 800 µl of lysis buffer preheated at 65°C.
Note: for some fungal strains, it is necessary to independenlty extract the DNA in several tubes. In this case, pool the independent DNA extractions in order to obtain a suitable amount of DNA. 

Mix the sample by using a Tissue Lyser at speed 6 m/s for 1 min and incubate at 65°C for 30 min.

SECTION: Bacterial and fungal lysis
4. Bacterial lysis

If growing bacteria in Nutrient Broth, centrifuge the bacterial culture at 5000g for 10 min, discard the supernatant and resuspend the bacterial pellet in preheated 800 µL of lysis buffer by pipetting vigorously. 

If growing the bacteria on Nutrient Agar plates, pick few isolated bacterial colonies (take the equivalent of two toothpick tips) and mix them into the preheated 800 µl of lysis buffer. 

For both methods, transfer the obtained suspension into the 2ml Eppendorf tube containing the glass-beads. Mix the solution and incubate at 65°C for 30 min.
 
SECTION: Bacterial and fungal lysis
6. Common steps to both bacteria and fungi
- Add 60 µL of 20% SDS stock. 
- Homogenize the samples by using a Tissue Lyser at speed 6 m/s for 1 min.
- Add 20 µl of lysozyme 100 mg/ml and incubate 30 min at 37°C.
- Add 8 µl of proteinase K and incubate overnight at 56°C.

SECTION: Protein precipitation (common to both bacteria and fungi)
7. Centrifuge 1 min at 10,000 x g.
SECTION: Protein precipitation (common to both bacteria and fungi)
8. Transfer the supernatant to a new tube (~ 650 µL) containing 200 µL of 5M potassium acetate.
SECTION: Protein precipitation (common to both bacteria and fungi)
9. Mix and incubate on ice for 5 min.
SECTION: Protein precipitation (common to both bacteria and fungi)
10. Centrifuge at maximum speed for 2 min. Transfer the supernatant to a new tube (~ 500 µl).
SECTION: Genomic DNA purification (common to both bacteria and fungi)
11. Add 350 µL of chloroform:Isoamyl alcohol (24:1).
SECTION: Genomic DNA purification (common to both bacteria and fungi)
12. Invert the tubes several times until the solution is homogenous and centrifuge 5 min at maximum speed.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
13. Take upper phase into a new tube (~ 300 µl) without disturb the inner phase. 
Note: if the upper phase contains traces of the microbial suspension, repeat the “chloroform:isoamyl alcohol” step twice.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
14. Mix the cleaned aqueous phase with an equal volume of isopropanol and a 1/8 volume of 3M sodium acetate. 
Note: DNA will precipitate and form “strings”. 
SECTION: Genomic DNA purification (common to both bacteria and fungi)
15. Incubate the samples on ice for 10 min.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
16. Centrifuge at maximum speed for 5 min to obtain a DNA pellet.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
17. Discard the supernatant by reversing the tubes and let the samples dry for 5 min under the hood.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
18. Wash the pellet with 70% of ethanol and add 600 µl of 70% ethanol, centrifuge 5 min at maximum speed, discard the supernatant by reversing tubes and let the samples dry for 5 min under the hood. 
SECTION: Genomic DNA purification (common to both bacteria and fungi)
19. Resuspend the pellet in 200 µl of Elution Buffer and add 900 µl of beads buffer.
Note: This will purify DNA and remove small DNA fragments.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
20. Incubate on a rotator at 10 rpm for 45 min at room temperature.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
21. Spin down the tubes for 1 second.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
22. Place the tubes on a magnetic rack and wait for 5 min (until solution becomes clear).
SECTION: Genomic DNA purification (common to both bacteria and fungi)
23. Discard the supernatant by pipetting without disturbing the beads.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
24. Add 70% ethanol to the tube. 
Note: Beads must be completely immersed in ethanol.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
25. Flick the tubes to resuspend beads, spin down and put the tubes back on the magnetic rack. Remove the ethanol without disturbing the beads.
SECTION: Genomic DNA purification (common to both bacteria and fungi)
26. Repeat the steps 24 to 25 and remove the ethanol with a P200 / P10 pipette. 
SECTION: Genomic DNA purification (common to both bacteria and fungi)
27. Let the samples air drying on the magnetic rack for 5 min.
Note: Beads must not dry too much otherwise resuspension can be difficult. 
SECTION: Genomic DNA elution (common to both bacteria and fungi)
28. Add 100 µl of Elution Buffer. 
SECTION: Genomic DNA elution (common to both bacteria and fungi)
29. Resuspend the beads by gentling pipetting and spin down the tube for 1 second.
Note: Make sure the beads are not aggregated.
SECTION: Genomic DNA elution (common to both bacteria and fungi)
30. Incubate the tubes for 15 min at 37°C.
SECTION: Genomic DNA elution (common to both bacteria and fungi)
31. Spin down the tubes for 1 second.
SECTION: Genomic DNA elution (common to both bacteria and fungi)
32. Place the tubes on the magnetic rack and wait until the beads are well anchored and the solution is clear. 
SECTION: Genomic DNA elution (common to both bacteria and fungi)
33. Transfer the solution, consisting in the HMW DNA, into a 1.5ml Eppendorf tube.
SECTION: RNase treatment (common to both bacteria and fungi)
34. Add 5 µl of 100 mg/ml RNase A.
SECTION: RNase treatment (common to both bacteria and fungi)
35. Incubate at 50°C for 15 min.
SECTION: DNA quality control (common to both bacteria and fungi)
36. Determine the DNA concentration with NanoDrop‱ or Qubit‱ Fluorometer. 
A260/280 ratio should ideally be between 1.8 and 2.
A260/230 ratio should ideally be between 2 and 2.2.
SECTION: DNA quality control (common to both bacteria and fungi)
37. To check that DNA is not degraded or contaminated with RNA, migrate the DNA samples on 1.5% agarose gel.
SECTION: Example of bacterial and fungal DNA quality control
38. 
 

 

 


All DNAs have been successfully sequenced with the PacBio Sequel II HiFi technology.
SECTION: Bacterial and fungal lysis
3. Before to start:
i) Fill 2mL Eppendorf tubes with 0.5 mL of 1 mm glass beads
ii) Heat the lysis buffer for ≥ 30 min at 65°C before use