Sep 11, 2024
Rapid Bacterial Isolate Whole Genome Sequencing
- Adela Alcolea-Medina1,
- Luke Blagdon Snell1,
- CIDR RESEARCH2
- 1King's College London;
- 2Guy's & St. Thomas' NHS Foundation Trust.
Protocol Citation: Adela Alcolea-Medina, Luke Blagdon Snell, CIDR RESEARCH 2024. Rapid Bacterial Isolate Whole Genome Sequencing . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32ndev25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 05, 2024
Last Modified: September 11, 2024
Protocol Integer ID: 106992
Abstract
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
Set-up and sample sheet
Set-up and sample sheet
Table for sample information
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.
Isolate DNA extraction (bead-beating and bead wash):
Isolate DNA extraction (bead-beating and bead wash):
Bead-beating/mechanical lysis of bacterial isolates:
For each isolate, add 500uL PBS to a Lysing Matrix E 2mL (MP Biomedicals) bead-beating tube including one for negative control.
For each culture plate, take an entire quadrant using a 10uL loop and add to a bead-beating tube.
Start bead-beating at 1x40s 4m/s.
Centrifuge at 14,000 RPM for 5 minutes.
Bead wash
Transfer 150uL of supernatant from each Lysing Matrix E tube to new 1.5mL DNA Lobind Tube, one per sample.
Add 75uL of AMPure XP beads to each tube
Mix by flicking and incubate at RT for 5 minutes with frequent agitation.
Pulse spin briefly.
Place tubes magnetic rack for 3 minutes for beads to pellet.
Wash twice with 80% ethanol whilst keeping tube on magnetic rack.
Pulse spin briefly.
Dry the beads for 1 minute.
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.
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.
Library preparation and sequencing:
Library preparation and sequencing:
Library preparation for SQK-RBK114.96:
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.
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.
Incubate the tubes on a thermocycler at 30°C for 2 minute and 80°C for 2 minute*.
Pulse spin briefly.
Pool all barcoded samples together in a 1.5mL Lobind tube
Add equal amount of AMPure XP beads.
Mix by flicking till homogenise.
Incubate on hula-mixer for 5 minutes.
Pulse spin briefly.
Place on magnetic rack for 3 minute.
Remove supernatant.
Wash twice with 80% ethanol whilst keeping tube on magnetic rack.
Pulse spin briefly.
Remove remaining ethanol and air dry for 1 minute.
Remove tube from magnetic rack, add 11uL EB to elute the DNA
Incubate for 5 minutes on the hula-mixer.
Place on magnetic rack for 1 minute
Transfer 10uL of eluate into a fresh 1.5mL DNA Lobind Tube to give the final library.
Use 1uL of final library from the original tube for Qubit measurement
Wait for 2 minutes, and record Qbit concentration of final library (found on page 4).
Create a RA + ADB mix: Add 1.5ul of RA to 3.5ul of ADB.
Add 1ml of RA+ADB mix to 10ml of final library.
Mix by flicking and pulse spin briefly
Incubate the mixture for 5 minutes at RT on the hula-mixer. Move to library sequencing
steps 1-2 during incubation
In the tube, add as follows:
37.5uL Sequencing Buffer (SB)
25.5uL Library Beads (LIB)
11uL DNA library
Library sequencing:
Library sequencing:
Begin flowcell check on GridION for selected flowcell.
Record the number of pores.
Slide open the flow cell priming port and draw back a small volume to remove bubbles.
Add 800uL of flush buffer through the priming port
Wait for 5 minutes.
Open SpotON sample port cover.
Load 200uL of the flush buffer into the flow cell priming port (not the SpotON port).
Flick prepared library gently.
Add dropwise 75uL of the prepared library through the SpotON sample port.
Close all the ports and click start sequencing.
Input the run name in the following way:
<yymmdd>_<operator>_<isolate_run-number> and for sample name
<operator>_<isolate_run-number>.
Select the correct library kit: SQK-RBK114.96
Do not change any other settings and ensure FASTQ files are kept