Targeted Amplification sequencing of wastewater sample
A total of 10 mL of wastewater were concentrated using Nanotrap Magnetic Virus Particles (Ceres Nano) and total nucleic acids were extracted using Maxwell(R) HT Environmental TNA kits (Promega). Both steps were performed on a KingFisher Apex and Flex (ThermoFisher Scientic), respectively. The sequencing library was generated using QIAseq DIRECT SARS-CoV-2 Enhanced kits with the primer Booster (Qiagen) following manufacturer's instructions. Briefly, single-stranded viral RNA molecules were reverse transcribed into cDNA using hexaprimers. SARS-CoV-2 genome was then specifically enriched using a SARS-CoV-2 primer panel. The panel consists of approximately 550 primers for creating 425 amplicons, covering the entire SARS-CoV-2 viral genome. The second PCR was performed using seven cycles. The library preparation was fully automated using the Biomek i5 Automated Workstation (Beckman Coulter). Prior to sequencing, the library was quantified by qPCR using the QIAseq Library Quant System kit (Qiagen) and the quality was assessed using the QIAxcel Advanced System (Qiagen). The library was sequenced on an Illumina MiSeq platform using MiSeq Reagent v2 (300 cycles) kits.