27. Select ‘DNA’ on the Qubit 3.0 Fluorometer and then ‘ds High
Sensitivity’ as the assay. Press ‘Read Standards’ to proceed;
insert tube containing standard #1 and press ‘Read standard’,
remove tube when read is complete, and insert tube containing
standard #2 and press ‘Read standard’. When complete, press
‘Run samples’, select the sample volume (1μL) and units
(ng/μL), insert a sample tube and press ‘Read tube’, remove
tube when reading is complete, and insert next sample, until all
are read.
28. If required, genome copies can be measured in the sputum
samples using the Artus
® M. tuberculosis RG PCR Kit (Qiagen), as per the manufacturer’s instructions.
3.2 DNA Shearing
1. Dilute samples to 3μg DNA/130μL using TE LOW in a DNA
LoBind tube. If total DNA concentration is lower than 3μg,
add human DNA to a final amount of 3μg (see Note 3).
2. Using a pipette tip, introduce 130μL of DNA sample to Covaris microTube directly through the presplit septa. Do not
introduce bubbles.
3. Secure the microTube in the tube holder and shear for 6 Â 60 s
(duty cycle 10%, intensity 5, and 200 cycles per burst with
frequency sweeping) (see Note 8).
4. Remove microTube from Covaris and spin briefly in microfuge.
5. Remove sample using a pipette tip and transfer into a new
1.5mL DNA LoBind tube.
3.3 Post-shearing
Sample Purification
1. Add 180μL of homogenous AMPure XP beads (see Note 9) to
each sheared sample tube (~130μL), mix by vortexing for 5 s,
and incubate at room temperature for 5 min.
2. Briefly spin to collect sample to bottom of the tube.
3. Place tube on magnetic stand and allow the solution to clear
(3–5 min).
4. Keep the tube on the magnetic stand and carefully remove the
cleared supernatant without disturbing the beads.
5. With the tube still in place on the magnetic stand, add 500μL
of 70% ethanol and incubate for 1 min or when solution clears.
Remove ethanol and wash and repeat.
6. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tube.
7. Dry samples on heat block at 37 ˚C for not more than 5 min
(see Note 10).
8. Add 52μL of nuclease-free water directly to bead pellet. Mix by
vortexing and briefly spin tube to collect contents. Incubate for
2 min at room temperature.
Direct WGS of M. tb’
467
Sensitivity’ as the assay. Press ‘Read Standards’ to proceed;
insert tube containing standard #1 and press ‘Read standard’,
remove tube when read is complete, and insert tube containing
standard #2 and press ‘Read standard’. When complete, press
‘Run samples’, select the sample volume (1μL) and units
(ng/μL), insert a sample tube and press ‘Read tube’, remove
tube when reading is complete, and insert next sample, until all
are read.
28. If required, genome copies can be measured in the sputum
samples using the Artus
® M. tuberculosis RG PCR Kit (Qiagen), as per the manufacturer’s instructions.
3.2 DNA Shearing
1. Dilute samples to 3μg DNA/130μL using TE LOW in a DNA
LoBind tube. If total DNA concentration is lower than 3μg,
add human DNA to a final amount of 3μg (see Note 3).
2. Using a pipette tip, introduce 130μL of DNA sample to Covaris microTube directly through the presplit septa. Do not
introduce bubbles.
3. Secure the microTube in the tube holder and shear for 6 Â 60 s
(duty cycle 10%, intensity 5, and 200 cycles per burst with
frequency sweeping) (see Note 8).
4. Remove microTube from Covaris and spin briefly in microfuge.
5. Remove sample using a pipette tip and transfer into a new
1.5mL DNA LoBind tube.
3.3 Post-shearing
Sample Purification
1. Add 180μL of homogenous AMPure XP beads (see Note 9) to
each sheared sample tube (~130μL), mix by vortexing for 5 s,
and incubate at room temperature for 5 min.
2. Briefly spin to collect sample to bottom of the tube.
3. Place tube on magnetic stand and allow the solution to clear
(3–5 min).
4. Keep the tube on the magnetic stand and carefully remove the
cleared supernatant without disturbing the beads.
5. With the tube still in place on the magnetic stand, add 500μL
of 70% ethanol and incubate for 1 min or when solution clears.
Remove ethanol and wash and repeat.
6. After second wash step use a P10 tip to remove any residual
ethanol from the bottom of the tube.
7. Dry samples on heat block at 37 ˚C for not more than 5 min
(see Note 10).
8. Add 52μL of nuclease-free water directly to bead pellet. Mix by
vortexing and briefly spin tube to collect contents. Incubate for
2 min at room temperature.
Direct WGS of M. tb’
467