TITLE: Bead Beating in Custom Buffer Followed by XP Bead Cleanup (NGS Workflow) 
AUTHORS: Jason D Limberis, Alina Nalyvayko, Janré Steyn, Jennifer Williams, Melanie Grobbelaar, Robin M Warren, john.metcalfe

[DESCRIPTION]
DNA extraction method for Mycobacterium tuberculosis from various sample types as described in:  "Insights into Mycobacterium tuberculosis DNA extraction for targeted deep sequencing using the Deeplex Myc-TB assay: Lessons for improved drug resistance diagnosis."

[BEFORE_START]
Prepare buffers

[STEPS]
SECTION: Prepare Buffers
1. 
ComponentAmount to addNaCl (5M)2 mLTris-HCl pH 8.3 (1M)1 mLEDTA pH 9.0 (0.5M)0.2 mLTriton X-1001 mLH2O95.8 mL
 

ComponentAmount for 100 mLTris-HCl 1M pH 81 mLEDTA 0.5M20 uL

SECTION: Extract DNA
3. Transfer the inactivated bacterial suspension to a new well-labeled Starsted screw cap tube containing ~ 250 µL of Mini-BeadBeater Zirconia-Silicate Beads,  

SECTION: Extract DNA
4. Bead beat the lysate at 6.5m/s for 45 s with2 min rest on ice between runs

SECTION: Extract DNA
6. Add 250 µL (1.2X volume) AMPure XP beads and mix by pipetting up and down ten times
SECTION: Extract DNA
5. Centrifuge at max speed 10000 rcf, 2 min and transfer the supernatant to a new well-labeled tube.
Take care not to transfer beads or cell debris.
SECTION: Extract DNA
8. Place on magnetic rack and wait for the solution to clear, ~ 2 min

SECTION: Extract DNA
7. Incubate at Room temperature for 5 min
SECTION: Extract DNA
9. Discard the supernatant and wash the beads twice with freshly prepared 70 % (v/v) ethanol without disrupting the beads

SECTION: Extract DNA
10. Dry the beads briefly, ~ 2 min. 
Tip: Remove residual EtOH with a p10 pipette

SECTION: Extract DNA
11. Immediately after the bead pellet becomes opaque, remove the tube from magnetic rack and resuspend in 20 µL of Low EDTA Tris Buffer. Ensure all beads are in solution.

SECTION: Extract DNA
12. Incubate at room temperature for 5 min

SECTION: Extract DNA
13. Place on magnetic rack, wait for the solution to become clear ~2 min, and transfer the eluted DNA to a new well-labeled tube

None. 
None. 
None. If working with MGIT tubes, inactivate for 60 min at 80 °C according to the South African National Health Laboratory Services (NHLS) facility standard operating procedures.

SECTION: Prepare Input
2. If required (i.e., to remove samples for processing outside a BSL-3), decontaminate MGIT culture in the original tube and media for 60 minat 80 °C according to the standard operating procedures of the South African National Health Laboratory Services (NHLS) facility or your facilities guidelines.

SECTION: Prepare Input
2.1.   Centrifuge at max speed for 10 min

SECTION: Prepare Input
2.2. Resuspend in 350 µL of Custom Triton Buffer

SECTION: Extract DNA
4.1. Repeat for a total of three bead beating cycles

SECTION: Extract DNA
18. Place on magnetic rack, wait for the solution to become clear ~2 min, and transfer the eluted DNA to a new well-labeled tube

SECTION: Extract DNA
17. Incubate at room temperature for 5 min

SECTION: Extract DNA
16. Immediately after the bead pellet becomes opaque, remove the tube from magnetic rack and resuspend in 20 µL of Low EDTA Tris Buffer. Ensure all beads are in solution.

SECTION: Extract DNA
15. Dry the beads briefly, ~ 2 min. 
Tip: Remove residual EtOH with a p10 pipette

SECTION: Extract DNA
14. Discard the supernatant and wash the beads twice with freshly prepared 70 % (v/v) ethanol without disrupting the beads

SECTION: Extract DNA
13. Place on magnetic rack and wait for the solution to clear, ~ 2 min

SECTION: Extract DNA
12. Incubate at Room temperature for 5 min
SECTION: Extract DNA
11. Add 250 µL (1.2X volume) AMPure XP beads and mix by pipetting up and down 10 times
SECTION: Extract DNA
10. Centrifuge at max speed 10000 rcf, 2 min and transfer the supernatant to a new well-labeled tube.
Take care not to transfer beads or cell debris.
SECTION: Extract DNA
9.1. Repeat for a total of three bead beating cycles

SECTION: Extract DNA
9. Bead beat the lysate at 6.5m/s for 45 s with2 min rest on ice between runs

SECTION: Extract DNA
8. Transfer the inactivated bacterial suspension to a new well-labeled Starsted screw cap tube containing ~ 250 µL of Mini-BeadBeater Zirconia-Silicate Beads,  

7. If required (i.e., to remove samples for processing outside a BSL-3), decontaminate the sample according to the standard operating procedures of your facility.
5. Vortex sample and 15 min 

4. Remove and discard the supernatant, and resuspend the pellet in  350 µL of Custom Triton Buffer
SECTION: Prepare Input
3. Incubate at Room temperature for 15 min

SECTION: Prepare Input
2. Add four volumes of 100mM dithiothreitol to the sputum sample and vortex for >30 s

6. If it is not possible to do the above in your laboratory.
Use BBL MycoPrepTM (BD) reagent to process the sample according to the manufacturer's instructions with the following modification: Resuspend the sediment in step 8 in the protocol in 350 µLof Custom Triton Buffer
SECTION: Prepare Buffers
2. Cut the bottom off a StarSted tube just below the inflection point.
Cut the tip off a P1000 tip, cut a wedge near the end, and wedge the prepared StarSted tube in it.

 
SECTION: Prepare Buffers
3. Fill a sterile container (e.g., a Petri dish) with   Zirconia-Silicate Beads and use the prepared scoop to distribute them into 1.5ml StarSted tubes.