2.2. DNA Extraction and 16S rRNA Gene Amplicon Sequencing

Water samples were filtered in volumes of 25 mL (wastewaters) or 200 mL (surface water and effluent) using sterile 0.22 µm polyvinylidene difluoride (PVDF)-membrane filters. Filters were stored at −80 °C until DNA extraction was performed. DNA was extracted using the DNeasy Power Water kit (Qiagen, Hilden, Germany) and quantified using the Qubit dsDNA BR (broad range) Assay kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. DNA was stored at −20 °C before subsequent analysis. Amplicon sequencing of the V3–V4 regions of the 16S rRNA gene was performed on an Illumina MiSeq (Illumina, San Diego, CA, USA). Libraries were prepared by using the Nextera XT DNA Library Preparation Kit following the 16S Metagenomic Sequencing Library Preparation protocol, according to the manufacturer’s instructions [24]: the V3-V4 regions of the 16S rRNA gene were amplified by the polymerase chain reaction (PCR) using Amplicon primers with overhang adaptors (16S Amplicon PCR Forward Primer = 5′ TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG 16S Amplicon PCR Reverse Primer = 5′ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). A second PCR was used for attaching indices and Illumina sequencing adapters using the Nextera^® XT Index Kit (Illumina, San Diego, CA, USA). Fragments were cleaned after each PCR using freshly prepared Ampure XP Beads (Beckman Coulter Genomics, Danvers, MA, USA). In total, 171 samples were successfully sequenced, resulting in an average of 50,734 reads per sample (Table 1). The sample with the lowest number of reads was a downstream sample (18,059 reads), and the sample with the highest number of reads came from the hospital (138,846 reads). Sequence data are available in the NCBI sequence read archive (SRA) under project numbers PRJNA668059 and PRJNA668064.

16S rRNA gene amplicon sequencing results. The number of samples obtained per location, as well as the number of reads (average, minimal and maximal) are shown together with the rarefied and non-rarefied amplicon sequence variants (ASV) and the resulting average detection limit at log ratio. Libraries were rarefied for statistical comparison to 18,059 reads per sample.

¹ The log ratio of the detection limit per sample is calculated by: log(1)—log(total number of reads).