Sep 13, 2024
ROBIN: A unified nanopore-based sequencing assay integrating real-time, intraoperative methylome classification and next-day comprehensive molecular brain tumour profiling for ultra-rapid tumour diagnostics
- Simon Deacon1,
- Inswasti Cahyani1,
- Matthew Loose1
- 1University of Nottingham
Protocol Citation: Simon Deacon, Inswasti Cahyani, Matthew Loose 2024. ROBIN: A unified nanopore-based sequencing assay integrating real-time, intraoperative methylome classification and next-day comprehensive molecular brain tumour profiling for ultra-rapid tumour diagnostics. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xepklqe/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
We use this protocol and it's working
Created: January 08, 2024
Last Modified: September 13, 2024
Protocol Integer ID: 93072
Funders Acknowledgement:
Pathological Society of Great Britain
British Neuropathological Society
Jean-Shanks Foundation
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This protocol will require prior approval by the users' Institutional Review Board (IRB), or equivalent ethics committee, depending on the source of tissue used.
Abstract
Background
Advances in our technological capacity to interrogate brain tumour biology has led to the ever-increasing use of genomic sequencing in routine diagnostic decision making. Presently, brain tumours are routinely classified based on their epigenetic signatures, leading to a paradigm shift in diagnostic pathways. Such testing can be performed so rapidly using nanopore sequencing that results can be provided intraoperatively. This information greatly improves upon the fidelity of smear diagnosis and can help surgeons tailor their approach, balancing the risks of surgery with the likely benefit. Nevertheless, full integrated diagnosis may require subsequent additional assays to detect pathognomonic somatic mutations and structural variants, thereby delaying the time to final diagnosis.
Methods
Here, we present ROBIN, a tool based upon PromethION nanopore sequencing technology that can provide both real-time, intraoperative methylome classification and next-day comprehensive molecular profiling within a single assay. ROBIN uniquely integrates three methylation classifiers 1–3 to improve diagnostic performance in the intraoperative setting.
Findings
We demonstrate classifier performance on 50 prospective intraoperative cases, achieving a diagnostic turnaround time under 2 hours and generating robust tumour classifications within minutes of sequencing. Furthermore, ROBIN can detect single nucleotide variants (SNVs), copy number variants (CNVs) and structural variants (SVs) in real-time, and is able to inform a complete integrated diagnosis within 24 hours. Classifier performance demonstrated concordance with final integrated diagnosis in 90% of prospective cases.
Interpretation
Nanopore sequencing can greatly improve upon the turnaround times for standard of care diagnostic testing, including sequencing, and is furthermore able to reliably provide clinically actionable intraoperative tumour classification.
Guidelines
This protocol will require prior approval by the users' Institutional Review Board (IRB), or equivalent ethics committee, depending on the source of tissue used.
Materials
1. Qiagen QIAamp Fast DNA Tissue Kit (#51404)
2. Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
3. AMPure XP SPRI Reagent (A63880)
4. PEGW buffer (10% PEG-8000/0.5M NaCl; filtered only)
5. 10 mM Tris-HCl pH 9.0
6. Qubit 1X dsDNA High Sensitivity (HS) Assay kit (Q33230 or Q33231)
7. Genomic DNA ScreenTape Analysis (5067-5365 and 5067-5366)
8. 1.5 ml Eppendorf DNA LoBind tubes (#0030108051)
9. 0.2 ml PCR tubes
10. PromethION flow cells R10.4.1
11. Flow cell wash kit (EXP-WSH004 - additional)
Before start
In order to minimise the turnaround time in the interoperative setting, prior to reciept of sample:
- On ice, premix 9 µL FDB buffer with 3 µL FRA (from ONT SQK-ULK114).
- Prepare 3 Qubit tubes with 199 µL 1x HS reagent, and store in a dark place.
- Label all Eppendorfs required.
- Thaw all required library reagents in fridge.
- Prepare a DNA extraction master mix (from Qiagen QIAamp Fast DNA Tissue Kit) and pipette 265 µL into each of the 3 Tissue Disruption Tubes:
| Reagents | Volume (ul) | |
| AVE | 600 | |
| VXL | 120 | |
| DX | 3 | |
| PK | 60 | |
| RNAase A | 12 | |
| Total | 265μl x 3 |
Tissue Selection
Tissue Selection
Open tissue pot in Category 3 MSC and weigh total mass on petri dish.
Dissect out representative tumour tissue weighing 5-25 mg , then place in Tissue Disruption Tube.
Note
Reserve a portion of the adjacent tissue for intraoperative smear.
Note
Ensure lysis mix is added to Tissue Disruption Tubes prior to receipt of tissue.
Repeat steps 1-2 twice more, if tissue quantity allows.
Note
Ideally, three extractions from different representative parts of the tumour are performed.
Prepare a smear slide, using tissue adjacent to samples 1 to 3 arranged in a Left-to-Right orientation, labelled α, β, γ.
Note
Tissue labelled α, β, γ on slide to avoid confusion with routine laboratory nomenclature and sample labelling.
Note
During following DNA extraction steps, Neuropathologist to report smear and advise on most suitable sample to take forward for library preparation.
DNA Extraction from Brain Tissue
DNA Extraction from Brain Tissue
Homogenize tissue using vortexer at full speed for 00:05:00 .
Note
Proceed with step 4 regardless of whether there is residual tissue visible or not.
Incubate in a thermomixer at 1000 rpm, 56°C, 00:10:00 .
Note
If the lysate is homogenous after step 6, proceed directly with step 7. If there is still residual tissue left after step 6, repeat steps 5 and 6 a single time.
Add 265 µL Buffer MVL and mix by pipetting or vortexing.
Apply the mixture from step 7 to the QIAamp Mini spin column and centrifuge for 00:01:00 . Place the spin column into a new 2 ml collection tube.
Note
All centrifugation steps should be performed at maximum speed (up to 20,000 x g). Unless otherwise stated, all centrifugation steps are performed for 30 seconds.
Add 500 µL Buffer AW1 to the spin column and centrifuge. Place the spin column into a new 2 ml collection tube.
Add 500 µL Buffer AW2 to the spin column and centrifuge. Place the spin column into a new 2 ml collection tube.
Centrifuge for 00:01:30 . Place the spin column into a clean 1.5 ml microcentrifuge tube.
1m 30s
Add 100 µL ATE directly onto the spin column membrane, incubate at room temperature for00:02:00 .
Centrifuge for 00:01:00 and store DNA On ice .
DNA shearing
DNA shearing
Measure DNA concentration using 1X Qubit High Sensitivity (HS) reagents.
Note
As DNA concentration may reach >100 ng/ul, dilute the sample 10-fold when measuring with Qubit HS.
Take 2-10 µg DNA and dilute to 100 µL with 10 millimolar (mM) Tris-HCl pH 9.0 in a 1.5 ml Eppendorf LoBind tube. Aim for a DNA concentration of 20-40 ng/µL
Shear DNA by slowly but steadily passing the sample 18 times through a 30G x 1/2 needle.
Note
Ensure not to aspirate air into the syringe.
Measure sheared DNA concentration again using Qubit HS.
Library Preparation
Library Preparation
In a 1.5 ml Eppendorf LoBind tube, mix 600 ng DNA with the following components from the SQKULK114 (ONT) kit On ice :
| A | B | |
| Reagents | Volume (ul) | |
| DNA (~600ng) | x | |
| 10mM Tris-HCl pH 9.0 | 73-x | |
| FDB buffer | 15 | |
| Total | 88.0 |
Note
Prior to experiment, prepare premixed Eppendorf with 9 µL FDB with 3 µL FRA.
Add the diluted FRA to the DNA mixture using a P200 low-retention filtered tip. Keep On ice at all times, stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution. Mix gently and thoroughly by pipetting.
Note
Total volume is 100 µL . Make sure sample is homogeneous and cold while mixing with FRA. The FRA enzyme is fast acting and we want to make sure it is distributed evenly through the DNA solution before reacting.
Transfer into a suitable 0.2 mL tube. In a thermocycler, incubate the sample as follows:
30 °C for 00:05:00
80 °C for 00:02:00
4 °C for 00:01:00
Room temperature for 00:01:00
Note
Ensure that the thermocycler is able to start immediately when sample is ready (i.e. lid temperature has been reached and set to 90 °C if adjustable).
9m
Add 1 µL Rapid Adapter (RA). Mix gently by pipetting, and spin down.
Incubate the reaction at Room temperature for 00:04:00 with rotation at 9-10 rpm.
Note
Warm a PromethION flow cell at Room temperature in readiness for sequencing.
4m
Library Clean-up
Library Clean-up
Add 100 µL of resuspended AMPure XP Beads to the reaction and mix by pipetting or flicking the tube.
Note
Ensure AMPure XP Beads are at Room temperature before use.
Incubate the reaction at Room temperature for 00:04:00 with rotation at 9-10 rpm.
4m
Spin down the sample and leave to pellet on a magnet rack for 00:03:00 . Pipette off the supernatant.
3m
Wash the beads by adding 250 µL PEGW buffer (10% PEG-8000/0.5M NaCl; filtered only). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet for 00:03:00 . Pipette off the supernatant.
3m
Spin down and place the tube back on the magnetic rack. Pipette off any residual supernatant.
Remove the tube from the magnetic rack and resuspend the pellet in 50 µL of 10mM Tris-HCl pH 9.0. Spin down.
Incubate at 37 °C for 00:05:00 .
Note
During incubation, mix flowcell priming reagents: 980 µL Flow Cell Flush (FCF) + 25 µL Flush Tether UL (FTU)
Prime the promethION flow cell with 500 µL of the priming mixture.
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 00:01:00 .
Remove and retain the eluate containing the DNA library into a 1.5 ml Eppendorf DNA LoBind tube.
Quantify with Qubit HS.
Note
For troubleshooting, it may be necessary to quantify the DNA library using other methods, e.g. Agilent TapeStation genomic DNA and/or Nanodrop spectrophotometer, to validate library quantity and quality. Consider reserving 2ul of library for retrospective testing.
Library Loading and Sequencing
Library Loading and Sequencing
In a new 1.5 ml Eppendorf LoBind tube, prepare the library for loading as follows and mix well using a pipette:
| Reagents | Volume | |
| Sequencing Buffer (SBU) | 100 | |
| Library Solution (LSU) | 10 | |
| DNA library | 45 | |
| 10mM Tris-HCl pH 9.0 | 45 | |
| Total | 200 |
Note
In order to save reagents, the total loading volume can be reduced to 150 µL as follows:
| A | B | |
| Reagents | Volume | |
| Sequencing Buffer (SBU) | 75 | |
| Library Solution (LSU) | 7.5 | |
| DNA library | 45 | |
| 10mM Tris-HCl pH 9.0 | 37.5 | |
| Total | 150 |
Prime the promethION flow cell with 500 µL of the priming mixture.
Load the library mix into the flow cell. Let tether for at least 00:05:00 prior to sequencing.
Note
It is recommended to protect the flow cell array from ambient light (covers supplied by ONT)