DNA extraction for long-read sequencing of bacteria
Eby Sim
Abstract
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.
Steps
General bacteria culture
Streak bacteria of interest onto their respective, optimum solid media and incubate plates at optimal growth conditions.
On the day of DNA extraction, observe the plate to ensure purity. Do not attempt extraction if different colony morphologies are observed.
DNA extraction
To a clean 2 mL Powerbead tube, add 300µL and 50µL .
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.
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.
Briefly vortex the Powerbead tube to mix reagents and bacterial biomass.
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 0h 2m 0s.
Remove Powerbead tubes from the Vortex adaptor and centrifuge at 10000x g.
Carefully aspirate 300µL of supernatant without disturbing the pellet or picking up beads. Slowly dispense entire volume into a 2 mL collection tube.
Add 100µL 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.
Incubate the tube On ice for 0h 6m 0s.
Centrifuge the tube at 10000x g.
Carefully aspirate 300µL of supernatant, without disturbing the pellet, and slowly dispense into a 2 mL collection tube.
Add 600µL and gently invert tube to mix. If there is liquid stuck on the underside of the lid after mixing, pulse centrifuge collect the liquid.
Slowly aspirate 700µL of the DNA mixture and dispense into a spin column and centrifuge at 10000x g. Discard the flow through.
Slowly aspirate the remaining 200µL and dispense into the same spin column (as step 15) and centrifuge at 10000x g. Discard the flow through and replace the collection tube with a new collection tube.
Add 300µL and centrifuge at 10000x g. Discard the flow through and replace the collection tube with a new collection tube.
Perform a dry centrifuge at 10000x g to remove residual ethanol. Replace collection tube with a new 1.5 mL Lo-Bind tube.
Add 50µL to the centre of the membrane. Incubate at Room temperature for 0h 3m 0s.
Centrifuge the spin column at 10000x g. Discard spin column
DNA quality control
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.
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.
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.
Store DNA at -80oC.
