TITLE: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses
AUTHORS: Aaron Topol, Marlene Wolfe, Brad White, Krista Wigginton, Alexandria Boehm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This process instruction describes the steps for pre-analytical processing of primary settled solids from wastewater treatment plants for downstream nucleic acid purification and quantification. Previous research has demonstrated that enveloped viral particles, such as SARS-CoV-2, associate with the solids in wastewater. Therefore, concentrating the solids in the sample and removing the water concentrates the viral particles as well and increases the sensitivity of the assay.</div><div class = "text-block">Bovine coronavirus vaccine (BCoV) is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition.</div><div class = "text-block">The dry weight of the sample is determined to account for sample variability between different treatment plants and between samples collected on different days and allows for the final quantification to be normalized to the precise quantity of solids in the sample. A dry weight conversion factor to adjust for the final amount of solids in the quantified sample is determined by measuring the difference in mass between the “wet” dewatered solid and after the sample is dried at 110°C overnight.</div><div class = "text-block">For long term storage, up to 50mL of the original primary settled solid sample is stored at 4°C and small aliquot of the dewatered solids is stored at -80°C.</div><div class = "text-block">This process instruction applies to sample dewatering, BCoV control spike in, homogenization, dry weight measurement, aliquoting and storage. </div></div>

[STEPS]
?. [Preparation]
Process fresh samples stored at  if possible. If analyzing a frozen sample, thaw at  overnight. Set centrifuge temperature to .Set oven temperature to .Remove an aliquot of BCoV from the freezer and thaw .
4 °C
4 °C
4 °C
110 °C
on ice
?. [Aliquot and Sample Storage]
Apply a label for each sample to a 50 mL conical.
?. [Dewater Sludge Samples by Centrifugation]
Label 50 mL conicals with the sample name.
?. [Dewater Sludge Samples by Centrifugation]
Invert the sludge sample bottle a few times to mix.
?. [Dewater Sludge Samples by Centrifugation]
In a biosafety cabinet, pipette or pour  of well mixed primary sludge sample to corresponding labeled 50 mL conical. If the total volume of a sample is less than 50 mL, bring the volume up to 50 mL with nuclease free water.
50 mL
?. [Dewater Sludge Samples by Centrifugation]
Bring the rotor to a biosafety cabinet and arrange the conicals in the rotor in a manner to ensure that it is balanced.
?. [Dewater Sludge Samples by Centrifugation]
Centrifuge samples at  for at least  at . Set the Centrifuge time to “Hold”.
Centrifuge: 24000 34
4 °C
?. [Dewater Sludge Samples by Centrifugation]
In a biosafety cabinet, aspirate supernatant into a liquid biohazard waste container.
?. [BCoV Spike-in and Sample Homogenization]
Apply a label to a new 50 mL conical for each sample and record the empty conical mass in grams.
?. [BCoV Spike-in and Sample Homogenization]
With a spatula (either plastic or metal), scoop a small aliquot of dewatered pellet (~0.75 g) into the new, pre-weighed 50 mL conical. Record the mass in grams of the aliquot in the conical. Set the rest of the pellet aside for dry weight measurement.
It is important to note the wet mass of solids in this conical as it is essential in resuspending the solids in buffer to the right concentration and back calculating the concentration of the gene targets per dry mass of solids.
?. [BCoV Spike-in and Sample Homogenization]
In a new 150 mL bottle, prepare a stock solution of DNA/RNA shield with BCoV spike-in by adding  rehydrated BCoV vaccine per  DNA/RNA shield. Mix well by inverting or gently shaking the bottle.
1.5 µl
1 mL
The concentration of BCoV target is approximately 500,000 copies / ml in this solution.
?. [BCoV Spike-in and Sample Homogenization]
75 mg dewatered solids per ml BCoV-spiked DNA/RNA shield was chosen during an exercise where we titrated the solids in the mixture (spiked with gRNA of SARS-CoV-2) to determine the maximum mass per volume where we did not see inhibition of the SARS-CoV-2 assays in digital PCR. You may want to repeat this titration with your samples to ensure it is appropriate for alleviating inhibition.
75 mg dewatered solids per ml BCoV-spiked DNA/RNA shield was chosen during an exercise where we titrated the solids in the mixture (spiked with gRNA of SARS-CoV-2) to determine the maximum mass per volume where we did not see inhibition of the SARS-CoV-2 assays in digital PCR. You may want to repeat this titration with your samples to ensure it is appropriate for alleviating inhibition.
?. [BCoV Spike-in and Sample Homogenization]
Add 5-10 grinding balls to each conical tube.
?. [BCoV Spike-in and Sample Homogenization]
Place the conicals in the adapters on the stage of the Geno/Grinder and tightly secure the clamp.
?. [BCoV Spike-in and Sample Homogenization]
Shake the conicals in the Geno/Grinder for  at 1,000 rpm to homogenize the sample.
?. [BCoV Spike-in and Sample Homogenization]
Briefly centrifuge the conicals for  at  to pull down the bubbles generated during homogenization. Sample is ready for RNA extraction using the Chemagic 360 (see accompanying protocol).
Centrifuge: 3500 34
A modification of this step may provide greater sensitivity. The modification entails centrifuging at a higher speed of   to pellet the solids and then extracting the RNA from the resultant supernatant.
?. [BCoV Spike-in and Sample Homogenization]
Save the remaining DNA/RNA shield with BCoV spike-in to use as template in Extraction Positive Control during RNA Extraction.
?. [Dry weight measurement]
Place aluminum weigh boats in oven to pre-heat to .
110 °C
?. [Dry weight measurement]
Weigh each pan and record the mass in grams of each empty pan.
?. [Dry weight measurement]
Using a spatula, scoop a generous amount of dewatered solid into the pan.
?. [Dry weight measurement]
Record the mass in grams of each pan with the wet solids.
?. [Dry weight measurement]
Place the dishes in the oven inside the biosafety cabinet and leave overnight to dry,
?. [Dry weight measurement]
The next day ( -  later), remove the pans from the oven and record the mass in grams.
The dry weight percent is calculated as: (100)*(1 - (wet mass of solids - dry mass of solids) / (wet mass of solids))This percentage is key for calculation of the concentration of the gene targets per mass dry weight.
?. [Dry weight measurement]
Dispose of pans in the red biohazard waste bin.
?. [Aliquot and Sample Storage]
Pour or pipette up to  of remaining primary settled solids sample and place at  for storage until it is confirmed that results of the analyses pass quality control.  Dewatered solids can be stored at -80 deg C. We recommend that you confirm the effects of storage on target quantification. We have noted that up to 7 d storage at 4 deg C has minimal affect on the RNA targets. However, freeze thaws may affect target quantification.
50 mL
4 °C
?. [Aliquot and Sample Storage]
Dispose of remaining sludge in 4 L Biohazard waste container prefilled with  of bleach. Then dispose of the sample bottles as biohazard waste.
400 mL