Introduction

This extraction protocol is recommended for use on fungi/yeast and was validated on Candida albicans, Candida tropicalis and Candida parapsilosis.

Using this method you can expect a yield of ~40 ng/µl for fungi/yeast inputs.

Following this extraction method, you can perform an 8-plex library preparation using the Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) - Rapid Barcoding Kit V14 (SQK-RBK114.24 or SQK-RBK114.96) protocol.

This manual extraction method is a column-based extraction using the NEB Monarch Genomic DNA Purification Kit.

Equipment and consumables

Materials

• 2ml of 1~x10^7 cfu/ml overnight culture or a full 10 µl inoculating loop from a plate colony

Consumables

• MetaPolyzyme (Sigma, MAC4L-5MG)
• TE buffer (Sigma, 8890-100ML)
• 1.5 ml Eppendorf DNA LoBind tubes
• Phosphate Buffered Saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
• Proteinase K (NEB, P8107AAVIAL)
• Monarch® RNase A (NEB, T3018-1)
• CTAB buffer (Promega, MC1411)
• 5 M sodium chloride solution (Sigma, S6546)
• Monarch® gDNA Elution Buffer (NEB, T3016-1)
• Monarch® gDNA Binding Buffer (NEB, T3014-1)
• Monarch® gDNA Purification Columns (NEB, T3017-1)
• Monarch® Collection Tubes II (NEB, T2018-1)
• Monarch® gDNA Wash Buffer (NEB, T3015-1)
• Agencourt AMPure XP beads (Beckman Coulter™ cat # A63881)
• Nuclease-free water (e.g. ThermoFisher, AM9937)
• Freshly prepared 80% ethanol in nuclease-free water
• Qubit™ Assay Tubes (Invitrogen, Q32856)
• Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)

Equipment

• Thermal cycler
• Eppendorf 5424 centrifuge (or equivalent)
• Vortex mixer
• Thermomixer
• Qubit fluorometer (or equivalent)
• Magnetic rack

Optional equipment

• Hula mixer (gentle rotator mixer)

Method

1. Prepare the metapolyzyme by reconstituting in TE buffer to 20 mg/ml.

TIP: We recommend preparing enzymes freshly on the day as enzymatic activity reduces over time once reconstituted.

2. Centrifuge 2ml of 1~x10^7 cfu/ml overnight culture at 12,000 x g for 1 minute in a 2ml Eppendorf DNA LoBInd tube or take a full 10 µl inoculating loop from a plate.

3. Remove the supernatant and resuspend the pellet in 500 µl PBS.

IMPORTANT: We do not recommend increasing the temperature of the next incubation as this may negatively impact DNA quality and sequencing data.

4. Incubate the sample at 80°C for 20 minutes.
This heated incubation is important for efficient cell lysis.

5. Centrifuge at 12,000 x g for 1 minute.

6. Remove the supernatant by pipetting and resuspend the pellet in 1 ml PBS.
Washing in PBS removes any potential inhibitors from the nutrient broth, as well as any free DNA that may be degraded.

7. Centrifuge at 12,000 x g for 1 minute.

8. Remove the supernatant and resuspend the pellet in 100 µl TE buffer (10 mM Tris pH 8 and 1 mM EDTA).

9. Add 10 µl metapolyzyme to the sample and mix by pulse vortexing.

10. Incubate at 37°C for 45 minutes at 1000 RPM in a thermomixer.
If a thermomixer is unavailable, the reaction can be incubated stationary and agitated by gently flicking for 10 seconds every two minutes.

11. Combine the following reagents with your sample in the 1.5 ml Eppendorf DNA LoBind tube, and mix by vortexing.

Reagent	Volume
Proteinase K	30 µl
RNase A	9 µl
Total (including your sample)	149 µl

Vortex mixing your sample before adding the CTAB buffer allows the enzymes to fully mix with the bacterial cells as the mixture can become viscous.

12. Add 200 µl of CTAB buffer and mix by vortexing.
Extractions performed using the CTAB buffer improves sequencing health and output compared to using NEB Lysis Buffer from the Monarch Genomic Purification Kit.

13. Incubate at 56°C for 30 minutes at 1000 RPM in a thermomixer.

14. Add 100 µl 5 M sodium chloride and centrifuge at 17,000 x g for 5 minutes.
The high salt concentration reduces DNA yield but it is essential to remove any inhibitors of the library preparation.

15. Retain 200 µl of the lysate, taking care to avoid the pellet.
It is cruicial to avoid the pellet when pipetting off the lysate.

INFO: The NEB Monarch Genomic DNA Purification Kit is used for gDNA extraction. The remainder of this method is written following the Genomic DNA Binding and Elution protocol with our recommended alterations.

16. Preheat the Monarch® gDNA Elution Buffer to 60°C for later use.

IMPORTANT: The ratio of binding buffer to lysate (2:1) is important for the column binding efficiency.
We recommend taking through 200 µl of lysate for use with 400 µl of binding buffer per sample.
To run more samples from the same isolate, the volumes can be increase in relation to the 2:1 ratio of buffer to lysate.

17. Combine the following reagents in a clean 1.5 ml Eppendorf DNA LoBind tube:

Reagent	Volume
Lysate from the previous step	200 µl
Monarch® gDNA Binding Buffer	400 µl
Total	600 µl

18. Ensure the reaction is thoroughly mixed by pipetting until the liquid is homogenous.
Thorough mixing is essential to ensure the DNA binds properly to the column and to homogenise the CTAB and Monarch® gDNA Binding Buffer which solidify on initial contact.

19. Transfer the reaction to a Monarch® gDNA purification column pre-inserted into a Monarch® collection tube, without touching the upper column area.

20. Close the tube and centrifuge for 3 minutes at 1,000 x g to bind the gDNA and then for 1 minute at maximum speed (>12,000 x g) to clear the membrane.
Do not empty or remove the collection tube from the centrifuge when changing settings.

21. Remove the gDNA purification column from the collection tube and discard the tube with the flow-through.

22. Transfer the gDNA purification column to a new collection tube and add 500 µl Monarch® gDNA Wash Buffer.

23. Close the collection tube and invert multiple times until the wash buffer reaches the cap.

IMPORTANT: Do NOT vortex.

24. Immediately centrifuge the collection tube for 1 minute at maximum speed (>12,000 x g). Remove the gDNA purification column from the collection tube to discard the flow-through.

25. Re-insert the gDNA purification column into the collection tube and add 500 µl gDNA wash buffer and close the tube.

26. Immediately centrifuge the collection tube for 1 minute at maximum speed (>12,000 x g). Remove the gDNA purification column from the collection tube to discard the flow-through.

27. After centifugation, ensure there is no liquid in the gDNA purification column above or below the silica membrane. If there is liquid, repeat the centrifugation until all liquid is removed.

IMPORTANT: Preheating the Monarch® gDNA Elution Buffer to 60°C is essential for maximum recovery of larger gDNA fragments.

28. Place the gDNA purification column in a DNase-free 1.5 ml microfuge tube and add 100 µl of preheated Monarch® gDNA Elution Buffer. Close the tube and incubate at room temperature for 1 minute.

TIP: If extractions are not yielding high enough concentrations of DNA for 200 ng per sample (<11 ng/µl), less gDNA elution buffer can be run through the column to increase the concentration.
Note: Elution of less than 100 µl results in lower total yield and elution of less than 50 µl is not recommended.

29. Centrifuge for 1 minute at maximum speed (>12,000 x g) to elute the gDNA into the 1.5 ml microfuge tube.
The gDNA purification column can be discarded.

TIP: After DNA extraction perform a 0.4X AMPure XP bead wash.
This wash is to remove inhibitors to sequencing and we have found this results in improved sequencing outputs when loading the same amount of DNA.

30. Resuspend the AMPure XP Beads by vortexing.

31. Prepare the DNA in nuclease-free water:

For each sample:
1. Transfer 80 µl of extracted DNA sample into a fresh 1.5 ml Eppendorf DNA LoBind tube
2. Adjust the volume to 100 µl with nuclease-free water
3. Mix thoroughly by flicking the tube to avoid unwanted shearing.
4. Spin down briefly in a microfuge
Note: Multiple gDNA extractions from the same culture can be pooled to make up 80 µl and washed together

32. Prepare 500 µl of fresh 80% ethanol in nuclease-free water.

33. Add 40 µl of resuspended AMPure XP beads to the sample and mix by flicking the tube.

34. Incubate at room temperature for 5 minutes.
Note: If available, incubate on a Hula mixer (rotator mixer).

35. Spin down and pellet on a magnetic rack for 2 minutes. Keep the tube on the magnet and pipette off the supernatant.

36. Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol in nuclease-free water without disturbing the pellet. Remove the ethanol using a pipette and discard.

37. Repeat the previous step.

38. Briefly spin down and pellet the beads on the magnet. Pipette off any residual ethanol and allow to dry for ~30 seconds but do not dry to the point of cracking.

39. Remove the tube from the magnet and resuspend the pellet in 25 µl Monarch® gDNA Elution Buffer. To retain longer fragments lengths, incubate at 60°C for 5 minutes.

40. Pellet the beads on a magnet until the eluate is clear and colourless for at least 1 minute.

41. Remove and retain 20 µl of eluate in a fresh 1.5 ml Eppendorf DNA LoBind tube.

CHECKPOINT: Quantify 1 µl of eluted sample using a Qubit fluorometer.

Expected yield is ~40 ng/µl

END OF STEP: Take forward 200 ng per sample into the library preparation step of the Nanopore-only Microbial Isolate Sequencing Solution (NO-MISS) - Rapid Barcoding Kit V14 (SQK-RBK114.24 or SQK-RBK114.96) end-to-end protocol.

Results

Data for extraction method yields coming soon.

Change log

Version	Change
v1	Initial publication