TITLE:   VIRUSHUNTER™   ARTIC SARS-CoV-2 Whole Genome Sequencing Assay
AUTHORS: Kendall Hoff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The VirusHunter ARTIC SARS-CoV-2 Whole Genome Sequencing Assay uses a modified version of the sample prep method developed by Josh Quick and the ARTIC Network using the same multiplex RT-PCR primers (</span><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye</span></a><span>). The protocol begins with RT-PCR using two different primer pools for each sample. The pools are then combined and the RT-PCR products are fragmented using DNase I to generate small fragments that can more efficiently hybridize to QuadCore DNA chips from Centrillion Technologies. Following hybridization, chips are stained, scanned with the Summit scanner, and analyzed using VirusHunter software. Analysis workflows are not described herein (check out our GitHub page for analysis software).</span></div><br/><div class = "text-block"><span>Initial results of v1 of this protocol have been reported here: </span><a href="https://pubs.acs.org/doi/abs/10.1021/acs.langmuir.0c02927" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://pubs.acs.org/doi/abs/10.1021/acs.langmuir.0c02927</span></a></div><br/><div class = "text-block">with improvements to the calling algorirthm reported here:</div><div class = "text-block"><a href="https://www.biorxiv.org/content/10.1101/2021.05.11.443659v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.biorxiv.org/content/10.1101/2021.05.11.443659v1</span></a></div><br/><div class = "text-block">This is an updated version of the protocol with improved coverage and accuracy. Expected coverage is >99% with >99.9% possible for high-quality samples. Expected accuracy is >99.9% with >99.99% possible for high-quality samples.</div></div>

[STEPS]
?. 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.
?. RTIn the PCR cabinet, add  of the 5xLunaSript RT SuperMix to PCR tubes or plates.Add  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:Step&nbsp;Temperature&nbsp;Time&nbsp;Annealing&nbsp;25°C&nbsp;2 minutes&nbsp;Reverse Transcription&nbsp;55°C&nbsp;60 minutes&nbsp;Heat Inactivation&nbsp;95°C&nbsp;1 minute&nbsp;Hold&nbsp;4°C&nbsp;&nbsp;&nbsp;
2.2 µl
8.8 µl
Step&nbsp;Temperature&nbsp;Time&nbsp;Annealing&nbsp;25°C&nbsp;2 minutes&nbsp;Reverse Transcription&nbsp;55°C&nbsp;60 minutes&nbsp;Heat Inactivation&nbsp;95°C&nbsp;1 minute&nbsp;Hold&nbsp;4°C&nbsp;&nbsp;&nbsp;
?. Set up for Fragmenation and HybridizationPreheat a hybridization oven toPrewarm two thermal cyclers to  and.Resuspend lyophilized DNase I (Sigma #D4263):Add of 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 -°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  of resuspended enzyme to of 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.
45 °C
37 °C
95 °C
2 mL
-20 °C
40 µl
20 mL
?. Hybridize fragmented PCR product to QuadCore™ MicroarrayTransfer  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 .Incubate at  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.
60 µl
45 °C
45 °C
?. Wash/StainPreheat a hybridization oven to Prepare staining master mixes. Store on ice, dark:SA Stain Reagent&nbsp;Volume (uL): total = 50µL&nbsp;2x Number of Samples + 20%Nuclease-free water&nbsp;37&nbsp;20x SSC&nbsp;12.5&nbsp;Cy3-Streptavidin (1mg/mL)&nbsp;0.5&nbsp;Anti-SA Stain Reagent&nbsp;Volume (uL): total = 50µL&nbsp;Number of Samples + 10%Nuclease-free water&nbsp;42.3&nbsp;10x PBS&nbsp;5&nbsp;BSA (20mg/mL)&nbsp;2.5&nbsp;Biotinylated Goat anti-SA&nbsp;0.2&nbsp;After hybridization, transfer the chip plate to a wash tray containing of Wash A ifor  at room temperature Transfer the chip plate to a wash tray containing of Wash B and transfer to   for .Pipette of SA Stain MM into desired wells of a staining tray. Transfer the chip plate to this tray. Incubate for  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 of Wash A for  at room temperature.Pipette of Anti-SA Stain MM into desired wells of a staining tray. Transfer the chip plate to this tray. Incubate for at room temperature, dark. Transfer the chip plate to a wash tray containing of Wash A for  at room temperature.Pipette of SA Stain MM into desired wells of a staining tray. Transfer the chip plate to this tray. Incubate for   at room temperature, dark. Transfer the chip plate to a wash tray containing of Scan Buffer for  at room temperature.
30 °C
SA Stain Reagent&nbsp;Volume (uL): total = 50µL&nbsp;2x Number of Samples + 20%Nuclease-free water&nbsp;37&nbsp;20x SSC&nbsp;12.5&nbsp;Cy3-Streptavidin (1mg/mL)&nbsp;0.5&nbsp;
Anti-SA Stain Reagent&nbsp;Volume (uL): total = 50µL&nbsp;Number of Samples + 10%Nuclease-free water&nbsp;42.3&nbsp;10x PBS&nbsp;5&nbsp;BSA (20mg/mL)&nbsp;2.5&nbsp;Biotinylated Goat anti-SA&nbsp;0.2&nbsp;
20 mL
20 mL
30 °C
50 µl
20 mL
50 µl
20 mL
50 µl
20 mL
?. Image on Summit™ InstrumentImage 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™ scannerAdd of 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."
2 mL
?. Analyze data using the VirusHunter™ software
?. Samples can be stored at   at this point. If so, the heat denaturation ()and snap cool steps above should be repeated for best results.
-20 °C
95 °C
?. PCRIn 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.Reagent&nbsp;Volume (µL): total = 25µL&nbsp;Number of Samples +10%&nbsp;Nuclease-free water&nbsp;8.85&nbsp;&nbsp;5x Q5 Reaction buffer&nbsp;5&nbsp;&nbsp;Biotin-11-dUTP (1mM)&nbsp;1.25&nbsp;&nbsp;25mM dNTPs&nbsp;0.4&nbsp;&nbsp;Primer Mix 1 or 2&nbsp;(10µM)&nbsp;4&nbsp;&nbsp;Q5 Hot Start DNA Polymerase&nbsp;0.5&nbsp;&nbsp;Sample&nbsp;5&nbsp;-&nbsp;Aliquot of each MM to desired wells on a 96-well plate or to PCR tubes in the PCR cabinet.Add of 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:Step&nbsp;Temperature&nbsp;Time&nbsp;Heat Activation&nbsp;95°C&nbsp;30 seconds&nbsp;40 Cycles&nbsp;95°C&nbsp;15 seconds&nbsp;63°C&nbsp;5 minutes&nbsp;&nbsp;Final Extension&nbsp;72°C&nbsp;2 minutes&nbsp;Hold&nbsp;4°C&nbsp;&nbsp;&nbsp;
Reagent&nbsp;Volume (µL): total = 25µL&nbsp;Number of Samples +10%&nbsp;Nuclease-free water&nbsp;8.85&nbsp;&nbsp;5x Q5 Reaction buffer&nbsp;5&nbsp;&nbsp;Biotin-11-dUTP (1mM)&nbsp;1.25&nbsp;&nbsp;25mM dNTPs&nbsp;0.4&nbsp;&nbsp;Primer Mix 1 or 2&nbsp;(10µM)&nbsp;4&nbsp;&nbsp;Q5 Hot Start DNA Polymerase&nbsp;0.5&nbsp;&nbsp;Sample&nbsp;5&nbsp;-&nbsp;
20 µl
5 µl
Step&nbsp;Temperature&nbsp;Time&nbsp;Heat Activation&nbsp;95°C&nbsp;30 seconds&nbsp;40 Cycles&nbsp;95°C&nbsp;15 seconds&nbsp;63°C&nbsp;5 minutes&nbsp;&nbsp;Final Extension&nbsp;72°C&nbsp;2 minutes&nbsp;Hold&nbsp;4°C&nbsp;&nbsp;&nbsp;
?. Fragment and HybridizationImportant! Fragmentation is highly time sensitive. A multichannel is highly recommended so that reactions can be started and stopped for all samples ~simultaneously.Combine of the Pool 1 + of 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  for Add of 500x DNase I to samples. Vortex and briefly centrifuge samples.Incubate for at Important! This step is highly time sensitive. Start your timer as soon as the enzyme is added to the samples.To stop reactions, add of 4x ARTIC Hybridization Buffer. Vortex and briefly samples.Incubate for at Snap cool samples on ice.Seal, vortex, and briefly centrifuge samples.
20 µl
20 µl
37 °C
40 µl
37 °C
27 µl
95 °C