Rneasy powersoil kit

A modified method described by Peccia et al. [8 (link)] and optimized by Johnson et al. [9 (link)] was used in order to extract the total RNA using the Qiagen RNeasy^® PowerSoil^® Kit, as per the manufacturer’s instructions (Qiagen, Hilden, Germany). Briefly, 100 mL of influent wastewater was spun down at 2500× g for 20 min, whereafter 2–5 mL of the resultant supernatant pellet was added to a 15 mL PowerBead^® Tube containing a lysis buffer to inactivate the virus and stabilize the viral RNA. Thereafter, the sample was homogenized and phase-separated using an equal volume of phenol/chloroform and the upper aqueous phase was transferred to a new 15 mL tube and mixed with the required buffers as supplied within the RNeasy PowerSoil kit. The RNA isolation sample was then transferred to the RNeasy JetStar Mini Column to elute out the bound RNA before centrifugation at 13,000× g for 15 min. The resultant pellet was dried and dissolved in a final volume of 50 µL of ribonuclease-free water. The quantity and quality of the total RNA was measured by spectrophotometry using the NanoDrop^® ND-1000 instrument (Nanodrop Technologies, Wilmington, NC, USA). In the absence of a surrogate, a clinical SARS-CoV-2 positive nasal swab sample with known viral copies was used to spike a wastewater sample in order to investigate the efficiency of the extraction method.

RNA was extracted from ~2 g of thawed sediment following the RNeasy PowerSoil kit (QIAGEN). Sediment was thawed and homogenized but still cold when added into the bead and lysis solution. Extracted RNA was DNase treated with the TURBO DNA-free kit (Invitrogen), and was followed by ribosomal RNA depletion using the bacterial version of the RiboMinus Transcriptome Isolation Kit (ThermoFisher Scientific). Quantity and quality of extracted nucleic acids were measured on a NanoDrop One spectrophotometer (ThermoFisher Scientific). The RNA samples were confirmed to be free of DNA contamination using a 2100 Bioanalyzer (Agilent). Library preparation of RNA for sequencing was prepared with the TruSeq RNA Library Prep v2 kit skipping the poly-A selection step (Illumina). The RNA was sequenced on one Illumina NovaSeq6000 S4 lane with a paired-end 2 × 150-bp setup at the Science for Life Laboratory, Stockholm.

DNA extraction for 16S rRNA gene amplification was performed using the PowerSoil DNA Isolation Kit (Qiagen). Total DNA for metagenome sequencing was obtained using an Advanced Soil DNA Kit (mCHIP). Total RNA was recovered from the 1‐month‐old culture using an RNeasy® PowerSoil® Kit (Qiagen). The kits mentioned above were used according to the manufacturer's protocols.
Libraries for metagenomic sequencing were generated using the NEB Next® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs) following the manufacturer's recommendations with the addition of index barcodes. For metatranscriptomic sequencing, the detailed methods involving the removal of rRNA and library construction are described in the Supplementary Methods. The metagenomic and metatranscriptomic libraries were sequenced on an Illumina Novaseq. 6000 platform (Illumina) and generated 150‐bp paired‐end reads.

DNA and RNA were extracted from thawed and homogenized sediment with the DNeasy PowerMax soil kit (Qiagen) and RNeasy PowerSoil kit (Qiagen), with 10 g and 2 g input material, respectively. Extracted RNA was DNase treated with the Turbo DNA-free kit (Invitrogen) and rRNA depletion using the RiboMinus transcriptome isolation kit (bacterial version, ThermoFisher Scientific). Multiplexed libraries were prepared with the ThruPLEX DNA-seq (Rubicon Genomics) and TruSeq RNA Library Prep v2 [Illumina, without the poly(A) selection step] kits for DNA and RNA, respectively. Paired-end 2- by 150-bp sequencing was conducted at the Science for Life Laboratory, Stockholm, Sweden, on the Illumina NovaSeq platform (DNA on one lane, NovaSeq6000 S2, and RNA on one lane, Illumina NovaSeq6000 S4). The sequencing yielded on average 41 million (range 32 to 52) and 82 million (range 74 to 89) read-pairs for each DNA and RNA sample, respectively.