Bainomugisa, A. et al., 2018. A complete nanopore-only assembly of an XDR Mycobacterium tuberculosis Beijing lineage strain identifies novel genetic variation in repetitive PE/PPE gene regions. Microbial Genomics, doi: https://doi.org/10.1101/256719

Materials

• 1 inoculation loop full of solid culture in Löwenstein–Jensen medium
• ThermoFisher PrepMan® Ultra Sample Preparation Reagent
• 96% and 70% ethanol
• TE buffer (pH 7.5)
• 3 M sodium acetate (pH 5.5)
• Agencourt AMPure XP beads
• Nuclease-free water
• 0.7 mm glass beads
• 1.5 ml Eppendorf DNA LoBind tubes
• BioSpec BeadBeater
• Vortex mixer
• Hula mixer (gentle rotator mixer)
• Incubator/heat block
• Microfuge
• Magnetic rack

Methods

1. Dispense 700 µl of the PrepMan Ultra reagent into each sterile tube containing a small quantity of glass beads. Make sure the level of beads is no more than 50% of liquid level.

2. Place one loop-full of culture into the tube with beads. Remove the culture from the loop by gently twirling the loop in the beads. Mix thoroughly.

3. Heat at 95°C for 15 minutes.

4. Shear the cells in a BioSpec BeadBeater for 3 pulses of 40 seconds at 6.0 m/s.

5. Centrifuge the tube contents for 10 mins at 13,000 rpm.

6. Transfer 450 µl of the supernatant into a clean 1.5 ml Eppendorf DNA LoBind tube.

7. Add 50 µl of 3 M sodium acetate (pH 5.5) to the tube.

8. Add 1 ml of ice-cold 96% ethanol, and mix. Incubate the tube at –20°C for 30–60 mins.

9. Centrifuge the tube at 13,000 rpm for 15 min. Remove the supernatant by pipetting, and discard.

10. Add 1 ml of 70% ethanol and incubate for 1 min. Remove and discard the supernatant without disturbing the pellet.

11. Dry the pellet at 55–65°C for 10–15 min, avoiding over-drying the pellet.

12. Resuspend the pellet in 70 µl of nuclease-free water, and mix thoroughly.

13. Centrifuge the tube at 5000 rpm for 1–2, minutes and pipette the supernatant (~40–50 µl) into a clean 1.5 ml Eppendorf DNA LoBind tube.

14. Add 0.7x of AMPure XP beads to your DNA sample, and mix by flicking the tube. Incubate for 10 mins on a Hula mixer at room temperature.

15. Spin down briefly and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant. Keeping the tube on the magnet, wash beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the 70% ethanol using a pipette and discard. Repeat this wash step once more.

16. Spin down the tube and place it back on the magnet. Pipette off any residual 70% ethanol. Briefly allow to dry for 30 sec.

17. Elute the DNA in 40 µl nuclease-free water.

Results

• Yield: 10 µg
• OD 260/280: 1.99
• OD 260/230: 1.87
• Fragment size:

Sequencing performance

Libraries were prepared using the Ligation Sequencing Kit:

• Read length profile