TITLE: DNA extraction for long-read sequencing of bacteria 
AUTHORS: Eby Sim

[DESCRIPTION]
Utilisation of long-read sequencing can reliably generate complete bacterial genomes. Here, we present a DNA extraction method which introduces minor modifications to the DNeasy® UltraClean® Microbial Kit (Qiagen) to generate DNA suitable for long-read sequencing. When sequenced, the extracted DNA should yield median read lengths greater than 7 kb. In addition, this protocol uses cultures growing on solid media as a starting point which will be useful for laboratories that do not routinely use liquid cultures.

[BEFORE_START]
Users should take note that this protocol uses both mechanical lysis and spin-columns for DNA extraction. This protocol should not be used if reads lengths of > 60 kb is desired. The following image shows the typical read length distribution obtained from this methodology.

  



[GUIDELINES]
Users are reminded to be very gentle and deliberate in pipetting as this could shear DNA. Usage of wide-bore pipette tips (if accessible) will be preferable. 

[STEPS]
SECTION: General bacteria culture
1. Streak bacteria of interest onto their respective, optimum solid media and incubate plates at optimal growth conditions.  
SECTION: General bacteria culture
2. On the day of DNA extraction, observe the plate to ensure purity. Do not attempt extraction if different colony morphologies are observed. 
SECTION: DNA extraction
3. To a clean 2 mL Powerbead tube, add 300 µL and 50 µL .


SECTION: DNA extraction
4. Take a 1 µL Inoculation loop and pick up 4 streaks from the first quadrant and dislodge the biomass into the Powerbead tube containing both the PowerBead solution and Solution SL. 

SECTION: DNA extraction
5. Using a fresh 1 µL Inoculation loop, pick up 4 streaks from the second quadrant and dislodge biomass into the Powerbead tube containing both the PowerBead solution and Solution SL.
SECTION: DNA extraction
6. Briefly vortex the Powerbead tube to mix reagents and bacterial biomass. 


SECTION: DNA extraction
7. Carefully affix the Vortex adaptor onto the Vortex-Genie 2 vortex and place Powerbead tubes horizontally, with the cap facing inwards onto the Vortex adaptor. Vortex at maximum speed for 2 min.

SECTION: DNA extraction
8. Remove Powerbead tubes from the Vortex adaptor and centrifuge at 10000 x g, 1 min. 

SECTION: DNA extraction
9. Carefully aspirate 300 µL of supernatant without disturbing the pellet or picking up beads. Slowly dispense entire volume into a 2 mL collection tube.

SECTION: DNA extraction
10. Add 100 µL and gently finger-flick the collection tube to mix. If there is liquid stuck on the underside of the lid after mixing, give the tube a quick wrist-flick to collect the liquid.

SECTION: DNA extraction
12. Centrifuge the tube at 10000 x g, 2 min.

SECTION: DNA extraction
13. Carefully aspirate 300 µL of supernatant, without disturbing the pellet, and slowly dispense into a 2 mL collection tube.
SECTION: DNA extraction
14. Add 600 µL and gently invert tube to mix. If there is liquid stuck on the underside of the lid after mixing, pulse centrifuge collect the liquid.

SECTION: DNA extraction
11.  Incubate the tube on ice for 6 min.
SECTION: DNA extraction
15. Slowly aspirate 700 µL of the DNA mixture and dispense into a spin column and centrifuge at 10000 x g, 30 s. Discard the flow through.

SECTION: DNA extraction
16. Slowly aspirate the remaining 200 µL and dispense into the same spin column (as step 15) and centrifuge at 10000 x g, 30 s. Discard the flow through and replace the collection tube with a new collection tube.

SECTION: DNA extraction
17. Add 300 µL and centrifuge at 10000 x g, 30 s. Discard the flow through and replace the collection tube with a new collection tube.


SECTION: DNA extraction
18. Perform a dry centrifuge at 10000 x g, 1 min to remove residual ethanol. Replace collection tube with a new 1.5 mL Lo-Bind tube.
SECTION: DNA extraction
19. Add 50 µL to the centre of the membrane. Incubate at  Room temperature for 3 min.

SECTION: DNA extraction
20. Centrifuge the spin column at  10000 x g, 30 s. Discard spin column
SECTION: DNA quality control
22. Assess the concentration of dsDNA on a fluorometer (Qubit or equivalent). Ensure that concentration and amount of extracted dsDNA meets the requirement of the sequencing technology.  
 

SECTION: DNA quality control
23. Assess the integrity of the extracted dsDNA via electrophoretic separation (either via a 0.6% (w/v) agarose gel electrophoresis or TapeStation) using an appropriately sized ladder. 


 


SECTION: DNA quality control
21. Assess quality on a Spectrophotometers (NanoDropTM (Thermofiseher) or equivalent). Key quality metrics are listed below. Samples that do not fall within this value should not be sent for long-read sequencing.  While nucleic acid concentration is also measured by the spectrophotometer, it is not a key parameter at this stage. 
 

SECTION: DNA quality control
24. Store DNA at -80oC.