TITLE: Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y, del156-157/R158G, del143-145, LPPA24S, S:477-505, and ORF1a Del 141-143) in settled solids using digital RT-PCR
AUTHORS: Bridgette  Hughes, Bradley J. White, Marlene  K. Wolfe, Alexandria B B Boehm

[DESCRIPTION]
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 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G,  del143-145, and LPPA24S) 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. 

Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. 

This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR

The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). 

Scope 
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. 

Publications
The following publications describe use of these assays:

M. K. Wolfe, B. Hughes, D. Duong, V. Chan-Herur, K. R. Wigginton, B. White, A. B. Boehm. 2022. Detection of SARS-CoV-2 variant Mu, Beta, Gamma, Lambda, Delta, Alpha, and Omicron in wastewater settled solids using mutation-specific assays is associated with regional detection of variants in clinical samples. Applied and Environmental Microbiology. 88(8), e00045-22.Link.

A. B. Boehm, B. Hughes, M. K. Wolfe, B. J. White, D. Duong, V. Chan-Herur. Regional replacement of SARS-CoV-2 variant Omicron BA.1 with BA.2 as observed through wastewater surveillance. Accepted toEnvironmental Science & Technology Letters.Link to preprint.

Kirby AE, Welsh RM, Marsh ZA, Yu AT, Vugia DJ, Boehm AB, Wolfe MK, White BJ, Matzinger SR, Wheeler A, Bankers L, Andresen K, Salatas C, NYDEP, Gregory DA, Johnson MC, Trujillo M, Kannoly S, Smyth DS, Dennehy JJ, Sapoval N, Ensor K, Treangen T, Stadler LB, Hopkins L. 2022. Notes from the Field: Early Evidence of the SARS-CoV-2 B.1.1.529 (Omicron) Variant in Community Wastewater - United States, November - December 2021.MMWR Morb Mortal Wkly Rep2022;71:103- 105. DOI:http://dx.doi.org/10.15585/mmwr.mm7103a5.

A. Yu, B. Hughes, M. Wolfe, T. Leon, D. Duong, A. Rabe, L. Kennedy, S. Ravuri, B. White, K. Wigginton, A. Boehm, D. Vugia. 2022. Estimating relative abundance of two SARS-CoV-2 variants through wastewater surveillance at two large metropolitan sites.Emerging Infectious Disease, 28(5), 940-947.Link.






[STEPS]
SECTION: Preparation
1. Retrieve all kit components from the One-Step RT-ddPCR advanced kit for probes from the -20 °C freezer and thaw the components on ice.

  


 


SECTION: Preparation
2. Retrieve ddPCR positive control aliquots (50 copies per uL gRNA) from the -80 °C freezer and thaw on ice

SECTION: Preparation
3. For re-running frozen plates only: 

Thaw the RNA storage plate on ice. 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.

SECTION: Preparation
4. Keep extracted RNA samples on ice or in a cold block from the freezer at all times.



SECTION: ddPCR master mix preparation
5. Prepare Master Mix

SECTION: ddPCR master mix preparation
5.1. Prepare a working stock of fresh nuclease free water by pouring from a 50 mL Ambion Nuclease Free water into a 5mL eppendorf tube.
SECTION: ddPCR master mix preparation
5.2. Store Master Mix components on ice as much as possible during the preparation process. 

SECTION: ddPCR master mix preparation
5.3. Briefly vortex One-Step RT supermix for Probes (Yellow Tube), Reverse Transcriptase (Orange Tube), and DTT (Grey Tube) to mix contents then briefly spin with the benchtop centrifuge.

  
SECTION: ddPCR master mix preparation
5.4. In a 2mL eppendorf tube, prepare the master mix according to the table for SARS-CoV-2 quantification with HV69-70,  E484K/N501Y, or del156-157/R158G. Store prepared master mix on ice.


 

Primer/Probes Mixes

 


 


 



 
 
 

 
 
 


 


 


Master Mix

 

SECTION: Transfer Master Mix and Samples to ddPCR plate
6. For each assay, remove a cold block from the freezer and place a new Bio-Rad ddPCR 96-well Plate in it.
 


SECTION: Transfer Master Mix and Samples to ddPCR plate
7. Using a new reagent reservoir, manually pipette 16.5 µL of the appropriate Master Mix to each well of each ddPCR Plate.
SECTION: Transfer Master Mix and Samples to ddPCR plate
8. Transfer samples to the following wells on the plate either manually or using a liquid handling robot such as the Agilent Bravo system:
Transfer 5.5 µL of samples from columns 1-10 of the RNA plate into columns 1-10 of the ddPCR plate.
Transfer 5.5 µLof the extraction controls from column 12 of the RNA plate into column 11 of the ddPCR plate.
Transfer 5.5 µL of NTC (water) into A-G of column 12.
Add  5.5 µL of ddPCR positive control to well H12 of the ddPCR plate (add manually even if using a liquid handler)
 

The plate layout should be: 
  

 

SECTION: Transfer Master Mix and Samples to ddPCR plate
9. 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.
 

SECTION: Transfer Master Mix and Samples to ddPCR plate
10. Align a PCR Plate Heat Seal Foil on top of the ddPCR plate with the red line facing up.
SECTION: Transfer Master Mix and Samples to ddPCR plate
11. Seal the plate by pressing the green “seal” button.
SECTION: Transfer Master Mix and Samples to ddPCR plate
12. Briefly vortex the plate using a bench top plate vortexer.

SECTION: Transfer Master Mix and Samples to ddPCR plate
13. Briefly spin down the plate in the bench top Axygen Plate Centrifuge.
SECTION: Transfer Master Mix and Samples to ddPCR plate
14. Store the ddPCR plate on a white cold block or on ice until droplet generation.

SECTION: ddPCR droplet generation
15. Generate Droplets:
SECTION: ddPCR droplet generation
15.1. On the AutoDG droplet generator: select “configure plate” and then press the blue arrow in the upper left corner to highlight all columns.

SECTION: ddPCR droplet generation
15.2. 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.

SECTION: ddPCR droplet generation
15.3. Place the sealed, vortexed, and centrifuged ddPCR plate in the Sample Plate position on the AutoDG deck.

SECTION: ddPCR droplet generation
15.4. Remove the AutoDG cooling block from the freezer and place it in the Droplet Plate slot on the AutoDG deck.

SECTION: ddPCR droplet generation
15.5. For the Droplet Plate: label a new Bio-Rad ddPCR™ 96-Well Plate  and place it in the Auto DG cooling block.

SECTION: ddPCR droplet generation
15.6. Click “Generate Droplets” to begin droplet generation. Droplet generation will take approximately 45 min.

  


SECTION: ddPCR droplet generation
15.7. 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.


 

SECTION: ddPCR droplet generation
15.8. Align a PCR Plate Heat Seal Foil on top of the droplet plate with the red line facing up.
 

SECTION: ddPCR droplet generation
15.9. Seal the plate by pressing the green “seal” button.

 

SECTION: Thermocycling
16. Place the plate in the thermocycler. Verify and run the thermocycler program according to the table for SARS-CoV-2 ddPCR. 
 

  

 


 



SECTION: Thermocycling
16.1. After the PCR program is done, store the ddPCR plate at 4 °C (in the thermal cycler, on ice or in the fridge) until droplet reading.

SECTION: Read Droplets
17. 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.
SECTION: Read Droplets
18. 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. 


 

SECTION: Read Droplets
19. Name the file with a name clearly describing which plate you are reading.
SECTION: Read Droplets
20. 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.
SECTION: Read Droplets
21. 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”.
SECTION: Post-Processing Analysis
22. SARS-CoV-2 triplex assay analysis
SECTION: Post-Processing Analysis
22.1. Open QuantaSoft™ Analysis Pro Software
SECTION: Post-Processing Analysis
22.2. Open the .qlp file associated with the run

SECTION: Post-Processing Analysis
22.3. 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”:

 
  

SECTION: Post-Processing Analysis
22.4. 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”.
SECTION: Post-Processing Analysis
22.5. If wells need to be deselected: Go to the Plate Editor tab, select wells to exclude and click “Clear Selected Well”.
SECTION: Post-Processing Analysis
22.6. 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

  

SECTION: Post-Processing Analysis
22.7. Go through every well to ensure that:
The droplets are designated properly per well
There are >10,000 droplets per well 
SECTION: Post-Processing Analysis
22.8. If any well does not have the appropriate droplets, go to the Plate Editor tab, select the affected well and click “Clear Selected Well”.
SECTION: Post-Processing Analysis
22.9. On the 2D Amplitude tab, select Merged Wells on the left side of the page

SECTION: Post-Processing Analysis
22.10. Click on the Table Menu Button on the right side of the Well Data table and select Export to CSV to export data. 

SECTION: Post-Processing Analysis
22.11. Save the QuantaSoft Analysis
SECTION: Dimensional Analysis and Quality Control
23. For dimensional analysis to express the results of each assay in terms of gene copies/dry weight solids:
SECTION: Dimensional Analysis and Quality Control
23.1. Begin with the concentration provided by the QuantaSoft software and reported in the CSV, as expressed in gc/uL of reaction.
 

SECTION: Dimensional Analysis and Quality Control
23.2. To determine the concentration in cp/g dry weight: