VIRUSHUNTER™ ARTIC SARS-CoV-2 Whole Genome Sequencing Assay

RNA Extraction: this protocol is for the sequencing of SARS-CoV-2 from previously positive patient samples. Leftover RNA from qPCR testing can be used for this assay assuming proper storage/handling.

RT

In the PCR cabinet, add 2.2µL of the 5xLunaSript RT SuperMix to PCR tubes or plates.

Add 8.8µL of your template RNA in a distinct sample addition cabinet. Gently mix and pulse spin.

Transfer your samples to a thermal cycler and incubate as follows:

A	B	C
Step	Temperature	Time
Annealing	25°C	2 minutes
Reverse Transcription	55°C	60 minutes
Heat Inactivation	95°C	1 minute
Hold	4°C

PCR

In the PCR cabinet, prepare two PCR master mixes (one with Pool 1 primers and one with Pool 2 primers) for each sample. Gently mix and pulse spin.

A	B	C
Reagent	Volume (µL): total = 25µL	Number of Samples +10%
Nuclease-free water	8.85
5x Q5 Reaction buffer	5
Biotin-11-dUTP (1mM)	1.25
25mM dNTPs	0.4
Primer Mix 1 or 2 (10µM)	4
Q5 Hot Start DNA Polymerase	0.5
Sample	5	-

Aliquot 20µLof each MM to desired wells on a 96-well plate or to PCR tubes in the PCR cabinet.

Add 5µLof cDNA to the designated wells in the sample addition cabinet.

Do this once for the Pool 1 MM and once for the Pool 2 MM.

Transfer samples to a thermal cycler and incubate using the following program:

A	B	C
Step	Temperature	Time
Heat Activation	95°C	30 seconds
40 Cycles	95°C	15 seconds
	63°C	5 minutes
Final Extension	72°C	2 minutes
Hold	4°C

Set up for Fragmenation and Hybridization

Preheat a hybridization oven to45°C

Prewarm two thermal cyclers to 37°C and95°C.

Resuspend lyophilized DNase I (Sigma #D4263):

Add 2mLof ice cold 1x DNase I Buffer to a vial of lyophilized DNase I.

Important! To avoid loss of material when opening the vial, gently tap the bottom of the vial before opening. Open

slowly and be certain not to touch the stopper to the bench.

Reseal the vial with the stopper and invert 10 times to thoroughly mix.

Incubate for 30 minutes on ice to ensure full resuspension.

Invert an additional 10 times.

Note: store aliquots of resuspended enzyme at --20°C°C for up to 3 days. Do not re-freeze aliquots after thawing. Discard enzyme after use.

Prepare a fresh 500x dilution of DNase I using room temperature 1x DNase I buffer:

Add 40µL of resuspended enzyme to 20mLof room temperature 1xDNase I buffer.

Invert ten times to mix. Pour into a reagent reservoir.

Prepare 4x Hybridization buffer for use with a multichannel pipette - either aliquot to PCR tubes or add to a reagent reservoir.

Fragment and Hybridization

Important! Fragmentation is highly time sensitive. A multichannel is highly recommended so that reactions can be started and stopped for all samples ~simultaneously.

Combine 20µLof the Pool 1 + 20µLof the Pool 2 RT-PCR products for each of the starting samples in a new PCR tube or plate.

Ensure samples are sealed. Gently vortex and spin.

Prewarm samples at 37°C for 0h 5m 0s

Add 40µLof 500x DNase I to samples. Vortex and briefly centrifuge samples.

Incubate for 0h 45m 0sat 37°C

Important! This step is highly time sensitive. Start your timer as soon as the enzyme is added to the samples.

To stop reactions, add27µL of 4x ARTIC Hybridization Buffer. Vortex and briefly samples.

Incubate for0h 5m 0s at 95°C

Snap cool samples on ice.

Seal, vortex, and briefly centrifuge samples.

Samples can be stored at -20°C at this point. If so, the heat denaturation (95°C)and snap cool steps above should be repeated for best results.

Hybridize fragmented PCR product to QuadCore ™ Microarray

Transfer 60µL of each sample to the desired wells of the hybridization tray. 

Important! The well capacity of the 96-well hybridization tray is 40-70µL.

Hybridization relies on the target concentration in solution, not the total volume. If the full sample volume cannot be transferred without avoiding bubbles, do not attempt to transfer the full volume.

Optional: fill surrounding wells with 4xSSC to prevent evaporation.

Transfer the QuadCore™ chip plate to the tray and transfer the plate to 45°C.

Incubate at 45°C for 2 hours or overnight.

Note: longer incubation times of 16-24 hours will provide higher quality scores for base calling, however shorter incubation times of 1-6 hours can be used.

Wash/Stain

Preheat a hybridization oven to 30°C

Prepare staining master mixes. Store on ice, dark:

A	B	C
SA Stain Reagent	Volume (uL): total = 50µL	2x Number of Samples + 20%
Nuclease-free water	37
20x SSC	12.5
Cy3-Streptavidin (1mg/mL)	0.5

A	B	C
Anti-SA Stain Reagent	Volume (uL): total = 50µL	Number of Samples + 10%
Nuclease-free water	42.3
10x PBS	5
BSA (20mg/mL)	2.5
Biotinylated Goat anti-SA	0.2

After hybridization, transfer the chip plate to a wash tray containing of Wash A ifor at room temperature 20mLof Wash A ifor 0h 1m 0s at room temperature

Transfer the chip plate to a wash tray containing 20mLof Wash B and transfer to 30°C for 0h 5m 0s.

Pipette 50µLof SA Stain MM into desired wells of a staining tray. Transfer the chip plate to this tray. 

Incubate for 0h 2m 0s at room temperature, dark. 

Note: staining may be improved by the use of an orbital shaker.

Transfer the chip plate to a wash tray containing 20mLof Wash A for 0h 2m 0s at room temperature.

Pipette 50µLof Anti-SA Stain MM into desired wells of a staining tray. Transfer the chip plate to this tray. 

Incubate for 0h 10m 0sat room temperature, dark. 

Transfer the chip plate to a wash tray containing 20mLof Wash A for 0h 2m 0s at room temperature.

Pipette 50µLof SA Stain MM into desired wells of a staining tray. Transfer the chip plate to this tray. 

Incubate for  0h 2m 0s at room temperature, dark. 

Transfer the chip plate to a wash tray containing 20mLof Scan Buffer for 0h 1m 0s at room temperature.

Image on Summit ™ Instrument

Image on the Centrillion Technologies Summit™ Scanner equipped with the VirusHunter™ software, using three acquisition times (0.25, 1, and 4 seconds)

Important! QuadCore™MicroarraysProcessed withthe ARTIC-SARS-CoV-2-01 protocol will yield the best results if imaged over multiple acquisition times.

Initialize the software: double click the “Magpie” icon on the desktop to run the magpie program.

Select the location to store the files, and press “OK” button。

Right-Click the “Magpie” icon on the right lower of desktop and select the “E-Tools” option.

Open the lid of the Summit Scanner and place the scan tray into the machine. Ensure that the tray is flat and secure. 

Important: Never insert a wet scan tray into the Summit™ scanner

Add 2mLof Scan Buffer to the scan tray.

Important! Avoid buffer leakage in the Summit™ at all costs! By adding buffer to the scan tray after it is in the holder, you limit the chance of leakage during the placement of the scan tray in the holder. Do not overfill the scan tray.

Add your plate with the RFID facing away.

Important! Be very careful to insert the plate in the correct orientation with the RFID in the back and the notch at the front left corner. The “Scanning Tray” label should be visible.

Close the lid.

Click the "plate in" button to enable reading of the RFID.

Select your scanning channels and your chip numbers.

If you would like to have preview open while scanning, click preview before starting the scan.

Click "Scan."

Analyze data using the VirusHunter™ software