TITLE: Rapid Bacterial Isolate Whole Genome Sequencing 
AUTHORS: Adela Alcolea-Medina, Luke Blagdon Snell, CIDR RESEARCH

[DESCRIPTION]
Please cite the original publication where this process was described: Charalampous, T., Alcolea-Medina, A., Snell, L.B. et al. Evaluating the potential for respiratory metagenomics to improve treatment of secondary infection and detection of nosocomial transmission on expanded COVID-19 intensive care units. Genome Med 13, 182 (2021). https://doi.org/10.1186/s13073-021-00991-y

[STEPS]
SECTION: Set-up and sample sheet
1. 
 

SECTION: Set-up and sample sheet
1.1. Before beginning:
Ensure that culture purity plate has been left for 48 hours in the incubator and colonies have 
matured based on the amount of growth.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
2. Bead-beating/mechanical lysis of bacterial isolates:
SECTION: Isolate DNA extraction (bead-beating and bead wash):
2.1. For each isolate, add 500uL PBS to a Lysing Matrix E 2mL (MP Biomedicals) bead-beating tube including one for negative control.

SECTION: Isolate DNA extraction (bead-beating and bead wash):
2.2. For each culture plate, take an entire quadrant using a 10uL loop and add to a bead-beating tube.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
2.3.  Start bead-beating at 1x40s 4m/s.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
2.4.  Centrifuge at 14,000 RPM for 5 minutes.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3. Bead wash
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.1. Transfer 150uL of supernatant from each Lysing Matrix E tube to new 1.5mL DNA Lobind Tube, one per sample.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.2.  Add 75uL of AMPure XP beads to each tube
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.3. Mix by flicking and incubate at RT for 5 minutes with frequent agitation.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.4. Pulse spin briefly.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.5.  Place tubes magnetic rack for 3 minutes for beads to pellet.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.6.  Wash twice with 80% ethanol whilst keeping tube on magnetic rack.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.7.  Pulse spin briefly.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.8.  Dry the beads for 1 minute.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.9. Take each tube from the magnetic rack, add 50uL of PBS to each tube, and allow DNA to elute during 5 minute incubation at room temperature.
SECTION: Isolate DNA extraction (bead-beating and bead wash):
3.10. Place each tube back on the magnetic rack, allow beads to pellet, and retrieve 35uL to 50uL of the eluted DNA into a new 1.5mL DNA Lobind Tube.
SECTION: Library preparation and sequencing:
4. Library preparation for SQK-RBK114.96:
SECTION: Library preparation and sequencing:
4.1. Qubit the samples and record the amount of DNA (found on page 4) with the BroadRange/BR Qubit reaction kit. Use 2uL of sample into 198uL of Qubit mastermix.
SECTION: Library preparation and sequencing:
4.2. Add 7.5uL of the extracted DNA for each sample to 2.5uL of RBK114.96 barcodes in 0.2mL PCR tubes, one unique barcode per sample.
SECTION: Library preparation and sequencing:
4.3.  Incubate the tubes on a thermocycler at 30°C for 2 minute and 80°C for 2 minute*.
SECTION: Library preparation and sequencing:
4.4.  Pulse spin briefly.
SECTION: Library preparation and sequencing:
4.5.  Pool all barcoded samples together in a 1.5mL Lobind tube
SECTION: Library preparation and sequencing:
4.6. Add equal amount of AMPure XP beads.
SECTION: Library preparation and sequencing:
4.7.  Mix by flicking till homogenise.
SECTION: Library preparation and sequencing:
4.8.  Incubate on hula-mixer for 5 minutes.
SECTION: Library preparation and sequencing:
4.9.  Pulse spin briefly.
SECTION: Library preparation and sequencing:
4.10.  Place on magnetic rack for 3 minute.
SECTION: Library preparation and sequencing:
4.11.  Remove supernatant.
SECTION: Library preparation and sequencing:
4.12. Wash twice with 80% ethanol whilst keeping tube on magnetic rack.
SECTION: Library preparation and sequencing:
4.13.  Pulse spin briefly.
SECTION: Library preparation and sequencing:
4.14. Remove remaining ethanol and air dry for 1 minute.
SECTION: Library preparation and sequencing:
4.15. Remove tube from magnetic rack, add 11uL EB to elute the DNA
SECTION: Library preparation and sequencing:
5. Incubate for 5 minutes on the hula-mixer.
SECTION: Library preparation and sequencing:
6. Place on magnetic rack for 1 minute
SECTION: Library preparation and sequencing:
7. Transfer 10uL of eluate into a fresh 1.5mL DNA Lobind Tube to give the final library.
SECTION: Library preparation and sequencing:
8. Use 1uL of final library from the original tube for Qubit measurement
SECTION: Library preparation and sequencing:
9. Wait for 2 minutes, and record Qbit concentration of final library (found on page 4). 
SECTION: Library preparation and sequencing:
11. Add 1ml of RA+ADB mix to 10ml of final library.
SECTION: Library preparation and sequencing:
12. Mix by flicking and pulse spin briefly
SECTION: Library preparation and sequencing:
13. Incubate the mixture for 5 minutes at RT on the hula-mixer. Move to library sequencing
steps 1-2 during incubation
SECTION: Library preparation and sequencing:
14. In the tube, add as follows:
   37.5uL Sequencing Buffer (SB)
   25.5uL Library Beads (LIB)
   11uL DNA library
SECTION: Library preparation and sequencing:
15. 
 

SECTION: Library sequencing:
16. Begin flowcell check on GridION for selected flowcell.
SECTION: Library sequencing:
17. Record the number of pores.
SECTION: Library sequencing:
18. Slide open the flow cell priming port and draw back a small volume to remove bubbles.
SECTION: Library sequencing:
19. Add 800uL of flush buffer through the priming port
SECTION: Library sequencing:
20. Wait for 5 minutes.
SECTION: Library sequencing:
21. Open SpotON sample port cover.
SECTION: Library sequencing:
22. Load 200uL of the flush buffer into the flow cell priming port (not the SpotON port).
SECTION: Library sequencing:
23. Flick prepared library gently.
SECTION: Library sequencing:
24. Add dropwise 75uL of the prepared library through the SpotON sample port.
SECTION: Library sequencing:
25. Close all the ports and click start sequencing.
SECTION: Library sequencing:
26. Input the run name in the following way:
<yymmdd>_<operator>_<isolate_run-number> and for sample name
<operator>_<isolate_run-number>.
SECTION: Library sequencing:
27. Select the correct library kit: SQK-RBK114.96
SECTION: Library sequencing:
28. Do not change any other settings and ensure FASTQ files are kept
SECTION: Library preparation and sequencing:
10. Create a RA + ADB mix: Add 1.5ul of RA to 3.5ul of ADB.