TITLE: High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR
AUTHORS: Aaron Topol (Verily Life Sciences), marlene.wolfe , Brad White (Verily Life Sciences), 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 ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) 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><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>. </span></div><div class = "text-block">This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). </div><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 (both assays)]
Retrieve the ddPCR One-Step RT Supermix for probes from the  freezer and thaw the components .
-20 °C
on ice
?. [Preparation (both assays)]
Retrieve ddPCR positive control aliquots (50 copies per uL gRNA and 100 copies per µL BCoV and PMMoV gene blocks) from the  freezer and thaw
-80 °C
on ice
?. [Preparation (both assays)]
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 (both assays)]
Keep extracted RNA samples  or in a cold block from the freezer at all times.
on ice
IMPORTANT NOTE: The PMMoV assay is very sensitive to contamination due to the high copy number of PMMoV RNA molecules in the lab. It is essential to always use freshly prepared water and other reagents when setting up the BCoV/PMMoV assay and the change gloves and wipe down the working space and pipettes frequently with clean water.
?. [ddPCR master mix preparation (SARS-CoV-2 targets)]
Prepare SARS-CoV-2 Target Master Mix
This is a triplex assay for the N, S, and ORF1a gene. It is possible to swap out one or two of the assays for others that also target low copy number targets. 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 (SARS-CoV-2 targets)]
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 (SARS-CoV-2 targets)]
Store Master Mix components  as much as possible during the preparation process.
on ice
?. [ddPCR master mix preparation (SARS-CoV-2 targets)]
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 (SARS-CoV-2 targets)]
In a 2mL eppendorf tube, prepare the master mix according to the table for SARS-CoV-2 quantification. Store prepared master mix on ice.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
The 20x SARS-CoV-2 Primer/Probe Mix contains primers and probes in the following concentrations (suspended in molecular grade water):20x ConcentrationFinal Concentration/RxnPrimers (each)18 µM900 nMN Probe (FAM)5 µM250 nMS Probe (HEX)5 µM250 nMORF1a Probe (HEX)2.5 µM125 nMORF1a 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 (both assays)]
For each assay, remove a white 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.
?. [ddPCR droplet generation (both assays)]
Generate Droplets:
?. [ddPCR droplet generation (both assays)]
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 (both assays)]
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 red if the plates and tips are oriented correctly.
?. [ddPCR droplet generation (both assays)]
Place the sealed, vortexed, and centrifuged ddPCR plate in the Sample Plate position on the AutoDG deck.
?. [ddPCR droplet generation (both assays)]
Remove the AutoDG cooling block from the freezer and place it in the Droplet Plate slot on the AutoDG deck.
?. [ddPCR droplet generation (both assays)]
For the Droplet Plate: label a new Bio-Rad ddPCR™ 96-Well Plate  and place it in the black cooling block.
?. [ddPCR droplet generation (both assays)]
Click “Generate Droplets” to begin droplet generation. Droplet generation will take approximately .
During Droplet Generation for the SARS-CoV-2 plate, set up the PMMoV/BCoV plate.
?. [ddPCR droplet generation (both assays)]
When droplet generation is done, open the lid and remove the droplet plate from the black cold block and place it in the metal carrier inside the plate sealer.
?. [ddPCR droplet generation (both assays)]
Align a PCR Plate Heat Seal Foil on top of the droplet plate with the red line facing up.
?. [ddPCR droplet generation (both assays)]
Seal the plate by pressing the green “seal” button.
NOTE: Do NOT vortex plate at this point!
?. [Thermocycling (SARS-CoV-2)]
Place the plate in the thermocycler. Verify and run the thermocycler program according to the table for SARS-CoV-2 ddPCR.
?. [Thermocycling (SARS-CoV-2)]
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
?. [ddPCR master mix preparation (BCoV and PMMoV)]
Prepare Master Mix for the BCoV/PMMoV assay
Store Master Mix components on ice as much as possible during the preparation process.
?. [ddPCR master mix preparation (BCoV and PMMoV)]
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 (BCoV and PMMoV)]
In a 2mL eppendorf tube, prepare the master mix according to the table for PMMoV/BCoV quantification. Store prepared master mix on ice.ReagentsVolume per SampleVolume Per PlateddPCR™ One-Step RT supermix for Probes (Yellow Tube)5.5 µL580.8  µLBCoV/PMMoV Primer/Probe Mix1.1  µL232.32  µLReverse Transcriptase (Orange Tube)2.2  µL232.32  µL300mM DTT (Gray Tube)1.1 µL116.16 µLNuclease Free Water10.45 µL580.8 µLTotal Volume16.5  µL1742.4  µL
The 20x BCoV/PMMoV Primer/Probe Mix contains primers and probes in the following concentrations (suspended in molecular grade water):20x ConcentrationFinal Concentration/RxnPrimers (each)18 µM900 nMBCoV Probe (FAM)5 µM250 nMPMMoV Probe (HEX)5 µM250 nM
ReagentsVolume per SampleVolume Per PlateddPCR™ One-Step RT supermix for Probes (Yellow Tube)5.5 µL580.8  µLBCoV/PMMoV Primer/Probe Mix1.1  µL232.32  µLReverse Transcriptase (Orange Tube)2.2  µL232.32  µL300mM DTT (Gray Tube)1.1 µL116.16 µLNuclease Free Water10.45 µL580.8 µLTotal Volume16.5  µL1742.4  µL
?. [Sample storage]
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 (both assays)]
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 (both assays)]
Load template from previous plate of the same assay - ensure that all parameters (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.
?. [Read Droplets (both assays)]
Name the file with a name clearly describing which plate you are reading.
?. [Read Droplets (both assays)]
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 (both assays)]
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”.
?. [SARS-CoV-2 Post-Processing Analysis]
SARS-CoV-2 triplex assay analysis
?. [SARS-CoV-2 Post-Processing Analysis]
Open QuantaSoft™ Analysis Pro Software
?. [SARS-CoV-2 Post-Processing Analysis]
Open the .qlp file associated with the run
?. [SARS-CoV-2 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”:
?. [SARS-CoV-2 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”.
?. [SARS-CoV-2 Post-Processing Analysis]
If wells need to be deselected: Go to the Plate Editor tab, select wells to exclude and click “Clear Selected Well”.
?. [SARS-CoV-2 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
?. [SARS-CoV-2 Post-Processing Analysis]
Go through every well to ensure that:The droplets are designated properly per wellThere are >10,000 droplets per well
?. [SARS-CoV-2 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”.
?. [SARS-CoV-2 Post-Processing Analysis]
On the 2D Amplitude tab, select Merged Wells on the left side of the page
?. [SARS-CoV-2 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.
?. [SARS-CoV-2 Post-Processing Analysis]
Save the QuantaSoft Analysis
?. [BCoV/PMMoV post-processing analysis]
For BCoV and PMMoV analysis:
?. [BCoV/PMMoV post-processing analysis]
Open QuantaSoft™ Analysis Pro Software
?. [BCoV/PMMoV post-processing analysis]
Open the .qlp file associated with the run
?. [BCoV/PMMoV post-processing analysis]
On the Plate Editor tab, select all the wells then in the dropdown menu for Assay Information select Simplex/Duplex and complete as follows and click Apply:
?. [BCoV/PMMoV post-processing analysis]
.On the 1D Amplitude tab, make sure that all wells are highlighted and select the Threshold Multiple Wells button to automatically set the threshold between the positive and negative populationsBCoV threshold = 5000PMMoV threshold = 1700
?. [BCoV/PMMoV post-processing analysis]
Go through every well to ensure that:- The threshold accurately differentiates positive and negative droplets - There are >10,000 droplets per well
?. [BCoV/PMMoV 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”.
?. [BCoV/PMMoV post-processing analysis]
On the 1D Amplitude tab, select Merged Wells on the left side of the page
?. [BCoV/PMMoV 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. Save the CSV file.
?. [BCoV/PMMoV post-processing analysis]
Save the QuantSoft Analysis
?. [Sample storage]
Place the plate in the thermocycler. Verify and run the thermocycler program according to the table for BCOV/PMMoV ddPCR.
?. [ddPCR plate set up (BCoV and PMMoV)]
Dilute the RNA 1:100 for BCoV/PMMoV ddRT-PCR
The RNA must be diluted because of the high expected concentration of these targets.
The dilution of the RNA can be completed using a liquid handling robot. We use the Agilent Bravo system.
?. [ddPCR plate set up (BCoV and PMMoV)]
Using a new reagent reservoir, manually pipette  of nuclease free water into every well of an Eppendorf twin.tec PCR Plate labeled “100x RNA Dilution”.
198 µl
?. [ddPCR plate set up (BCoV and PMMoV)]
Manually pipette  of RNA from the stock RNA plate into each corresponding well in the dilution plate.
2 µl
?. [ddPCR plate set up (BCoV and PMMoV)]
Seal the plate and briefly vortex the plate using a bench top plate vortexer.
?. [ddPCR plate set up (BCoV and PMMoV)]
Briefly spin down the plate in the Axygen Plate Centrifuge.
?. [ddPCR plate set up (BCoV and PMMoV)]
Store plate until ready to add sample to ddPCR plate.
on ice
?. [Transfer Master Mix and Samples to ddPCR plate (both assays)]
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 (both assays)]
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
For SARS-CoV-2 PCR: Add SARS-CoV-2 genomic RNA For PMMoV/BCoV PCR: Add PMMoV/BCoV gene block positive control
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 (both assays)]
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 (both assays)]
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 (both assays)]
Seal the plate by pressing the green “seal” button.
?. [Transfer Master Mix and Samples to ddPCR plate (both assays)]
Briefly vortex the plate using a bench top plate vortexer.
?. [Transfer Master Mix and Samples to ddPCR plate (both assays)]
Briefly spin down the plate in the bench top Axygen Plate Centrifuge.
?. [Transfer Master Mix and Samples to ddPCR plate (both assays)]
Store the ddPCR plate on a white cold block or  until droplet generation.
on ice
?. [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.