TITLE: 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
AUTHORS: Simon Deacon, Inswasti Cahyani, Matthew Loose

[DESCRIPTION]
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. 

[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: 

 






















[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.

[STEPS]
SECTION: Tissue Selection
1. Open tissue pot in Category 3 MSC and weigh total mass on petri dish.
SECTION: DNA Extraction from Brain Tissue
5. Homogenize tissue using vortexer at full speed for 5 min.
 

SECTION: Tissue Selection
2. Dissect out representative tumour tissue weighing 5-25 mg, then place in Tissue Disruption Tube.
 

 

SECTION: Tissue Selection
4. Prepare a smear slide, using tissue adjacent to samples 1 to 3 arranged in a Left-to-Right orientation, labelled α, β, γ.
 

 

SECTION: Tissue Selection
3. Repeat steps 1-2 twice more, if tissue quantity allows.
 

SECTION: DNA Extraction from Brain Tissue
6. Incubate in a thermomixer at 1000 rpm, 10 min, 56 °C. 
 

SECTION: DNA Extraction from Brain Tissue
7. Add 265 µL Buffer MVL and mix by pipetting or vortexing.
SECTION: DNA Extraction from Brain Tissue
8. Apply the mixture from step 7 to the QIAamp Mini spin column and centrifuge for 1 min. Place the spin column into a new 2 ml collection tube.
 

SECTION: DNA Extraction from Brain Tissue
9. Add 500 µL Buffer AW1 to the spin column and centrifuge. Place the spin column into a new 2 ml collection tube.
SECTION: DNA Extraction from Brain Tissue
10. Add 500 µL Buffer AW2 to the spin column and centrifuge. Place the spin column into a new 2 ml collection tube.
SECTION: DNA shearing
14. Measure DNA concentration using 1X Qubit High Sensitivity (HS) reagents.
 

SECTION: DNA Extraction from Brain Tissue
11. Centrifuge for 90 s. Place the spin column into a clean 1.5 ml microcentrifuge tube.
SECTION: DNA Extraction from Brain Tissue
12. Add 100 µL ATE directly onto the spin column membrane, incubate at room temperature for2 min.
SECTION: DNA Extraction from Brain Tissue
13. Centrifuge for 1 min and store DNA on ice.
SECTION: DNA shearing
15. 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 
SECTION: DNA shearing
16. Shear DNA by slowly but steadily passing the sample 18 times through a 30G x 1/2 needle.
 

SECTION: DNA shearing
17. Measure sheared DNA concentration again using Qubit HS.
SECTION: Library Preparation
18. In a 1.5 ml Eppendorf LoBind tube, mix 600 ng DNA with the following components from the SQKULK114 (ONT) kit on ice:
 

 

SECTION: Library Preparation
19. 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. 
 

SECTION: Library Preparation
20. Transfer into a suitable 0.2 mL tube. In a thermocycler, incubate the sample as follows:
30 °Cfor 5 min
80 °C for 2 min
4 °C for 1 min
Room temperature  for 1 min 

 

SECTION: Library Preparation
21. Add 1 µL Rapid Adapter (RA). Mix gently by pipetting, and spin down.
SECTION: Library Preparation
22. Incubate the reaction at Room temperature for 4 min with rotation at 9-10 rpm.
 

SECTION: Library Clean-up
23. Add 100 µL of resuspended AMPure XP Beads to the reaction and mix by pipetting or flicking the tube.
 

SECTION: Library Clean-up
24. Incubate the reaction at Room temperature for 4 min with rotation at 9-10 rpm.

SECTION: Library Clean-up
25. Spin down the sample and leave to pellet on a magnet rack for 3 min . Pipette off the supernatant.

SECTION: Library Clean-up
26. 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 3 min. Pipette off the supernatant.

SECTION: Library Clean-up
27. Spin down and place the tube back on the magnetic rack. Pipette off any residual supernatant. 

SECTION: Library Clean-up
28. Remove the tube from the magnetic rack and resuspend the pellet in 50 µL of 10mM Tris-HCl pH 9.0. Spin down.

SECTION: Library Clean-up
29. Incubate at 37 °C for 5 min.
 

SECTION: Library Clean-up
30. Prime the promethION flow cell with 500 µL of the priming mixture.
SECTION: Library Clean-up
31. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 min.
SECTION: Library Clean-up
32. Remove and retain the eluate containing the DNA library into a 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Library Clean-up
33. Quantify with Qubit HS.
 

SECTION: Library Loading and Sequencing
33.1. In a new 1.5 ml Eppendorf LoBind tube, prepare the library for loading as follows and mix well using a pipette:
 

 


SECTION: Library Loading and Sequencing
34. Prime the promethION flow cell with 500 µL of the priming mixture.

SECTION: Library Loading and Sequencing
35. Load the library mix into the flow cell. Let tether for at least 5 min prior to sequencing.