TITLE: Quantification of SARS-CoV-2 variant mutations (HV69-70 and E484K/N501Y) in settled solids using digital RT-PCR
AUTHORS: Bridgette Hughes, Bradley J. White, Marlene K. Wolfe, Krista Wigginton, Alexandria Boehm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 2 mutation assays (one for HV69-70 and one for E484K/N501Y) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. </div><br/><div class = "text-block">Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. </div><br/><div class = "text-block"><span>This protocol uses RNA extracted using this protocol: </span><a href="https://www.protocols.io/view/high-throughput-rna-extraction-and-pcr-inhibitor-r-btyrnpv6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA</span></a><span>. That RNA is generated from samples subjected to pre-analytical steps outlined in: </span><a href="https://www.protocols.io/view/high-throughput-pre-analytical-processing-of-waste-btyqnpvw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses</span></a><span>. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol </span><a href="https://www.protocols.io/view/high-throughput-sars-cov-2-pmmov-and-bcov-quantifi-btywnpxe" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR</span></a></div><br/><div class = "text-block">The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). </div><br/><div class = "text-block">Scope </div><div class = "text-block">This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader. </div></div>

[STEPS]
?. [Preparation ]
Retrieve the ddPCR One-Step RT Supermix for probes from the  freezer and thaw the components .
-20 °C
on ice
?. [Preparation ]
Retrieve ddPCR positive control aliquots (50 copies per uL gRNA) from the  freezer and thaw
-80 °C
on ice
?. [Preparation ]
For re-running frozen plates only: Thaw the RNA storage plate . Before analysis of thawed frozen samples, ensure that the plate is adequately sealed and briefly vortex and centrifuge the plate to ensure the samples are well mixed.
on ice
?. [Preparation ]
Keep extracted RNA samples  or in a cold block from the freezer at all times.
on ice
?. [ddPCR master mix preparation]
Prepare Master Mix
This is a triplex assay for the N, S, and a mutation gene. Protocols targeting the HV69-70 deletion and E484K/N501 mutation are in this document, but it is possible to swap out one or two of the assays for others that also target low copy number targets using the same procedure. However, we do not recommend multiplexing with a high copy number target like PMMoV because a high copy number target assay would need to use diluted RNA as template.
?. [ddPCR master mix preparation]
Prepare a working stock of fresh nuclease free water by pouring from a 50 mL Ambion Nuclease Free water into a 5mL eppendorf tube.
?. [ddPCR master mix preparation]
Store Master Mix components  as much as possible during the preparation process.
on ice
?. [ddPCR master mix preparation]
Briefly vortex One-Step RT supermix for Probes (Yellow Tube) and Reverse Transcriptase (Orange Tube) to mix contents then briefly spin with the benchtop centrifuge.
?. [ddPCR master mix preparation]
In a 2mL eppendorf tube, prepare the master mix according to the table for SARS-CoV-2 quantification with HV69-70 or E484K/N501Y. Store prepared master mix on ice.20x ConcentrationFinal Concentration/RxnPrimers (each)18 µM900 nMN Probe (FAM)5 µM250 nMS Probe (HEX)5 µM250 nME484K/N501Y Probe (HEX)2.5 µM125 nME484K/N501Y Probe (FAM)2.5 µM125 nM20x ConcentrationFinal Concentration/RxnPrimers (each)18 µM900 nMN Probe (FAM)5 µM250 nMS Probe (HEX)5 µM250 nMHV69-70 Probe (HEX)2.5 µM125 nMHV69-70 Probe (FAM)2.5 µM125 nMReagentsVolume per WellVolume Per PlateddPCR™ One-Step RT supermix for Probes (Yellow Tube)5.5 µL580.8  µL20x SARS-CoV-2 Primer/Probe Mix3.3  µL348.48  µLReverse Transcriptase (Orange Tube)2.2  µL232.32  µL300mM DTT (Gray Tube)1.1 µL116.16 µLNuclease Free Water4.4 µL464.64 µLTotal Volume16.5  µL1742.4  µL
Additional protocols will be published for other mutations associated with SARS-CoV-2 variants.
20x ConcentrationFinal Concentration/RxnPrimers (each)18 µM900 nMN Probe (FAM)5 µM250 nMS Probe (HEX)5 µM250 nME484K/N501Y Probe (HEX)2.5 µM125 nME484K/N501Y Probe (FAM)2.5 µM125 nM
20x ConcentrationFinal Concentration/RxnPrimers (each)18 µM900 nMN Probe (FAM)5 µM250 nMS Probe (HEX)5 µM250 nMHV69-70 Probe (HEX)2.5 µM125 nMHV69-70 Probe (FAM)2.5 µM125 nM
ReagentsVolume per WellVolume Per PlateddPCR™ One-Step RT supermix for Probes (Yellow Tube)5.5 µL580.8  µL20x SARS-CoV-2 Primer/Probe Mix3.3  µL348.48  µLReverse Transcriptase (Orange Tube)2.2  µL232.32  µL300mM DTT (Gray Tube)1.1 µL116.16 µLNuclease Free Water4.4 µL464.64 µLTotal Volume16.5  µL1742.4  µL
?. [Transfer Master Mix and Samples to ddPCR plate]
For each assay, remove a cold block from the freezer and place a new Bio-Rad ddPCR 96-well Plate in it.
Note that these steps may be completed with a liquid handling robot. We use the BRAVO system.
?. [Transfer Master Mix and Samples to ddPCR plate]
Using a new reagent reservoir, manually pipette  of the appropriate Master Mix to each well of each ddPCR Plate.
16.5 µl
?. [Transfer Master Mix and Samples to ddPCR plate]
Transfer samples to the following wells on the plate either manually or using a liquid handling robot such as the Agilent Bravo system:Transfer  of samples from columns 1-10 of the RNA plate into columns 1-10 of the ddPCR plate.Transfer of the extraction controls from column 12 of the RNA plate into column 11 of the ddPCR plate.Transfer  of NTC (water) into A-G of column 12.Add   of ddPCR positive control to well H12 of the ddPCR plate (add manually even if using a liquid handler)The plate layout should be:
5.5 µl
5.5 µl
5.5 µl
5.5 µl
Use SARS-CoV-2 genomic RNA (ATCC® VR-1986D™) mixed 1:1 with appropriate mutation standard as ddPCR positive control (see materials section of the protocol). The extraction positive controls are generated during RNA extraction per this companion protocol: https://www.protocols.io/view/high-throughput-rna-extraction-and-pcr-inhibitor-r-btyrnpv6. In that protocol, the extraction positive control consists of SARS-CoV-2 genomic RNA that does not contain the mutations HV69-70 and E484K/N501Y or other mutation targets.
Note: The set up of this plate is slightly different from the set up of the 96 well extraction plate used in our companion protocol. Column 11 of this plate uses column 12 of the extraction plate. The lower right hand corner was purposely chosen for the placement of the positive controls.
?. [Transfer Master Mix and Samples to ddPCR plate]
Bring the BioRad ddPCR plate with Master Mix and samples to the Bio-Rad PX1 PCR Plate Sealer and place it in the metal carrier inside the plate sealer.
Do not store the metal carrier inside the plate sealer, as it will become warm and warm the samples during sealing.
?. [Transfer Master Mix and Samples to ddPCR plate]
Align a PCR Plate Heat Seal Foil on top of the ddPCR plate with the red line facing up.
?. [Transfer Master Mix and Samples to ddPCR plate]
Seal the plate by pressing the green “seal” button.
?. [Transfer Master Mix and Samples to ddPCR plate]
Briefly vortex the plate using a bench top plate vortexer.
?. [Transfer Master Mix and Samples to ddPCR plate]
Briefly spin down the plate in the bench top Axygen Plate Centrifuge.
?. [Transfer Master Mix and Samples to ddPCR plate]
Store the ddPCR plate on a white cold block or  until droplet generation.
on ice
?. [ddPCR droplet generation ]
Generate Droplets:
?. [ddPCR droplet generation ]
On the AutoDG droplet generator: select “configure plate” and then press the blue arrow in the upper left corner to highlight all columns.
?. [ddPCR droplet generation ]
Load 3 DG32 cartridge plates and 2 tip boxes with the lid removed onto the AutoDG deck. The icons on the AutoDG display will change from yellow to green if the plates and tips are oriented correctly.
?. [ddPCR droplet generation ]
Place the sealed, vortexed, and centrifuged ddPCR plate in the Sample Plate position on the AutoDG deck.
?. [ddPCR droplet generation ]
Remove the AutoDG cooling block from the freezer and place it in the Droplet Plate slot on the AutoDG deck.
?. [ddPCR droplet generation ]
For the Droplet Plate: label a new Bio-Rad ddPCR™ 96-Well Plate  and place it in the Auto DG cooling block.
?. [ddPCR droplet generation ]
Click “Generate Droplets” to begin droplet generation. Droplet generation will take approximately .
?. [ddPCR droplet generation ]
When droplet generation is done, open the lid and remove the droplet plate from the Auto DG cold block and place it in the metal carrier inside the plate sealer.
The droplets are highly unstable at this stage.  The droplets should not be left on the generator longer than they need to be.  Once thermocycled the droplets are more stable.
?. [ddPCR droplet generation ]
Align a PCR Plate Heat Seal Foil on top of the droplet plate with the red line facing up.
?. [ddPCR droplet generation ]
Seal the plate by pressing the green “seal” button.
NOTE: Do NOT vortex plate at this point!
?. [Thermocycling ]
Place the plate in the thermocycler. Verify and run the thermocycler program according to the table for SARS-CoV-2 ddPCR. StepsCycle #sTemp (°C)Time115060 min21955 min3409530 secs40591 min419810 min5-4holdStepsCycle #sTemp (°C)Time115060 min21955 min3409530 secs40611 min419810 min5-4hold
The annealing temperature for the multiplex assay for N, S, and E484K/N501Y is higher (61°C) than that for N, S, and HV69-70 (59°C). A higher annealing temperature was necessary to ensure high specificity for the E484K/N501Y assay, and we found that the N and S assays performed with the same amplitude/efficiency of reaction at 61°C as at 59°C. For additional mutation assays, see those protocols for thermocycling conditions and take note of differences in recommended annealing temperature.
StepsCycle #sTemp (°C)Time115060 min21955 min3409530 secs40591 min419810 min5-4hold
StepsCycle #sTemp (°C)Time115060 min21955 min3409530 secs40611 min419810 min5-4hold
?. [Thermocycling ]
After the PCR program is done, store the ddPCR plate at  (in the thermal cycler, on ice or in the fridge) until droplet reading.
4 °C
?. [Read Droplets]
Before running the plate on the QX200 droplet reader, open the drawer on the left side of the instrument and check that there is adequate droplet reader oil and that the waste bottle is not overly full.
?. [Read Droplets]
Load template from previous plate of the same assay - ensure that all parameters (supermix, dye, absorbance etc.) are correct for the assay used and that sample names are assigned to the appropriate wells. Save as with a new plate name.
Double check that the correct supermix label is used for the read and that the channels are labeled appropriately. These parameters can NOT be changed after you run the plate.
?. [Read Droplets]
Name the file with a name clearly describing which plate you are reading.
?. [Read Droplets]
Remove the plate from the thermal cycler and secure it in the droplet reader by placing it in the stage, placing the metal brace on top of it and pressing down the black plastic tabs on either side of the brace.
?. [Read Droplets]
Click “Run” in Quantasoft to commence droplet reading.Select “Ok” when the next dialog box comes up. Do not change the settings from “Columns” and “FAM/HEX”.
?. [Post-Processing Analysis]
SARS-CoV-2 triplex assay analysis
?. [Post-Processing Analysis]
Open QuantaSoft™ Analysis Pro Software
?. [Post-Processing Analysis]
Open the .qlp file associated with the run
?. [Post-Processing Analysis]
1.1.1.On the Plate Editor tab, select all the wells then in the dropdown menu for Assay Information select Probe Mix Triplex and complete as follows and click “Apply”:
?. [Post-Processing Analysis]
At this point, if sample names were not added at the start of the run, fill in the sample name by selecting the wells, typing in sample ID and click “Apply”.
?. [Post-Processing Analysis]
If wells need to be deselected: Go to the Plate Editor tab, select wells to exclude and click “Clear Selected Well”.
?. [Post-Processing Analysis]
On the 2D Amplitude tab, make sure that all wells are highlighted and select the Threshold Cluster Mode Graph Tool and draw 2D thresholds around each cluster as follows
?. [Post-Processing Analysis]
Go through every well to ensure that:The droplets are designated properly per wellThere are >10,000 droplets per well
?. [Post-Processing Analysis]
If any well does not have the appropriate droplets, go to the Plate Editor tab, select the affected well and click “Clear Selected Well”.
?. [Post-Processing Analysis]
On the 2D Amplitude tab, select Merged Wells on the left side of the page
?. [Post-Processing Analysis]
Click on the Table Menu Button on the right side of the Well Data table and select Export to CSV to export data.
?. [Post-Processing Analysis]
Save the QuantaSoft Analysis
?. [Dimensional Analysis and Quality Control]
For dimensional analysis to express the results of each assay in terms of gene copies/dry weight solids:
?. [Dimensional Analysis and Quality Control]
Begin with the concentration provided by the QuantaSoft software and reported in the CSV, as expressed in gc/uL of reaction.
This concentration is expressed in terms of the total volume of the merged wells, rather than the volume of the template added. Determination of gc/g relies on the ratio of of template in the reaction and mass in the volume eluted from extraction, so when beginning with this value expressed in terms of gc/uL the number of wells merged is not considered in the dimensional analysis.
?. [Dimensional Analysis and Quality Control]
To determine the concentration in cp/g dry weight:
When using the assays described in the three protocols that make up this process as written: B = 20 µL total reaction volumeA = 5 µL volume template in reactionC = 50 µL elution volume from each well in extraction kitZ = 0.0225 g mass of solids added to each extraction wellPercent solids varies for each sample on each day (typical range 0.05 - 0.3, i.e. 5-30%)Links to two  companion protocols to this protocol can be found in the description of this protocol.