Standard Operating Procedures for Interlaboratory and Methods Assessment of the SARS‐CoV‐2 Genetic Signal in Wastewater 2020 Introduction Interlaboratory and Methods Assessment of the SARS‐CoV‐2 Genetic Signal in Wastewater provides an assessment of the reliability and repeatability of laboratory methods currently being used to test for the genetic signal of SARS‐CoV‐2 in untreated wastewater. A number of the study’s participating labs agreed to publicly share their Standard Operating Procedures, and the ones provided here cover each of the eight major method groups described in the Environmental Science: Water Research & Technology manuscript about this project. Contents SOP 1.1 .......................................................................................................................................................... 1 SOP 1.2(H) ..................................................................................................................................................... 6 SOP 1S.1(H) ................................................................................................................................................. 20 SOP 1S.2H .................................................................................................................................................... 26 SOP 1S.3(H) ................................................................................................................................................. 31 SOP 2.1 ........................................................................................................................................................ 32 SOP 2.2 ........................................................................................................................................................ 64 SOP 2.3 ........................................................................................................................................................ 96 SOP 2S.1 .................................................................................................................................................... 125 SOP 2S.2 .................................................................................................................................................... 127 SOP 2S.5 .................................................................................................................................................... 135 SOP 2S.6 .................................................................................................................................................... 141 SOP 3.1 ...................................................................................................................................................... 147 SOP 3.2 ...................................................................................................................................................... 148 SOP 3.3 ...................................................................................................................................................... 149 SOP 3.4 ...................................................................................................................................................... 154 SOP 3.5 ...................................................................................................................................................... 155 SOP 3.6 ...................................................................................................................................................... 189 SOP 3S.1 .................................................................................................................................................... 192 SOP 3S.2H .................................................................................................................................................. 194 SOP 4.3 ...................................................................................................................................................... 202 SOP 4S.1(H) ............................................................................................................................................... 220 SOP 4S.2(H) ............................................................................................................................................... 230 SOP 4S.3 .................................................................................................................................................... 238 SOP 4S.4 .................................................................................................................................................... 239 SOP 4S.5H .................................................................................................................................................. 254 SOP 4S.8(H) ............................................................................................................................................... 300 SOPs 2S.3 and 4S.7 .................................................................................................................................... 302 SOP 1.1 Standard Operating Procedure Collection and Analysis Protocols for SARS-CoV-2 RT-qPCR Analysis from Wastewater (Revised 6-5-20) Collection: 1. For each influent sample, pipette 5 mL of influent into a 15 mL conical tube and store it at -80oC. Nothing is added to the samples these are just raw influent archives. Each sampling day there will be two samples, one from SJC and one from JW, so a total of two, 5mL influent samples per day will get archived. 2. Each sampling day, a total of six tubes of each sample will be preserved with DNA/RNA shield (Zymo Research Cat. R1100-250). So for two samples a total of 12 tubes will be prepared each day. a. The ratio of DNA/RNA shield to sample is 250 µL of sample to 750µL of RNA shield (1x) for each tube. First pipette the RNA shield into a sterile 2 mL screw-capped cryotubes then add the sample. Mix by inverting a couple of times. b. Store the cryotubes containing the influent mixed with the DNA/RNA shield in the -80oC revco freezer. 3. We will extract three tubes per sample leaving the other three in the freezer as back-ups. RNA Extraction: 1. For each sample three replicate extractions will be performed (i.e. three for the SJCe influent and three for the JWPCP influent). The Quick-RNA Fecal/Soil microbe microprep kit (Zymo Research Cat. R2040) is used for the RNA extractions. 2. For each extraction, take the tubes out of the -80oC freezer and thaw them in the BSC (class II B2) in Virology B lab. 3. Two mock extractions will be performed with each batch. One with extraction control RNA (in vitro transcribed AdV IPC RNA) and one without the extraction control RNA. a. The first will have 240µL of DEPC water and 750µL of RNA shield. The other 10µL will be carrier RNA (Qiagen Cat. 1068337). b. The second will have 235µL of DEPC water and 750µL of RNA shield. The remaining volume will be the carrier RNA (10µL) and the AdV IPC extraction control RNA (5µL). 4. For each tube, place the entire volume (about 1 mL) into a bead bashing tube from the Zymo Quick- RNA fecal/soil kit (cat. R2040). There should be eight tubes in total. 5. Bead beat on the FastPrep24 homogenizer for 40 seconds at 6m/s. 6. To get rid of the foam after bead beating, spin the samples down in the refrigerated microcentrifuge in the Virology B lab set at 4oC. Spin at >12,000 x g for 5-7 minutes. Note the Zymo protocol says to spin for one minute, this was found to be insufficient to get rid of the foam. 7. After centrifugation, remove the supernatant and place in a clean microfuge tube then add 5 µL of the in vitro transcribed AdV IPC RNA (approximately 104 copies/µL, usually a -7 dilution) to each sample tube and to the one mock extraction tube containing 230µL of DEPC water. This can be added directly after the sample is transferred to the new tube. a. The remaining mock extraction tube does not get the AdV IPC RNA and serves as another negative extraction control. 1 SOP 1.1 8. To the two mock extraction tubes (one with extraction control RNA and one without) add 10 µL of carrier RNA diluted to 1ug/ul (Qiagen cat. 1068337). 9. Proceed with the Zymo extraction as stated in the instructions. a. Note that in step 4 of the Zymo protocol it states to transfer 400µL of supernatant to a new tube. The extraction volume we have is more than 400µL (usually around 700µL). We pipette a fixed volume of 700µL and place it in a sterile 2 mL tube then add an equal volume of RNA binding buffer (700µL) from the Zymo kit as stated in the Zymo kit’s instruction notes. b. The total volume is processed through one Zymo-Spin IIICG Column as stated in step 5 of the Zymo protocol (around 1.4mL). This will require multiple spins to get the entire volume through. Collect and save each of the supernatants from the spins. Combine all of the supernatants in a sterile 15 mL conical tube and add one volume of ethanol (approximately 1.4 mL) as stated in step 6 of the protocol. c. The total volume of supernatant with ethanol is processed through a single new Zymo-spin IIICG Column in step 7 (about 2.8 mL). This will require multiple spins to get the entire volume through the column. d. Proceed with the Zymo protocol as stated from step 8 on. e. Add an additional centrifuge spin (2 minutes, >12,000 x g) for step 17 is done to ensure the column is dry prior to the final elution step. This equates to two, two-minute spins for this