Characterization of the microbial community of a river can provide various indications, such as its general state of health or the presence of contamination. Furthermore, the study of Bacteroidetes, which have a high degree of host specificity, can provide information on the species involved in any fecal contamination. We report the characterization of the bacterial community of the Lambro river (Italy) through the analysis of the 16S rRNA gene from 15 sampling points.

1. 400 ml water samples were taken from 15 different sampling points

2a. samples were filtered through a paper filter to eliminate soil and/or plant contaminants, and subsequently, the bacteria were collected on a 0.22 µm membrane. The bacteria were then resuspended by stirring the filters in 3 ml of sterile H2O for 1 hour, and a subsequent centrifugation at 12,000 rpm for 30 minutes collected a bacterial pellet. Bacterial DNA was extracted using the NucleoSpin Tissue kit (Qiagen) following the supplier’s directions. The DNA was resuspended in a final volume of 40 µl of sterile H20.

2b. The bacterial DNA from fecal samples, samples of human, dog, cat, and pig was obtained using the NucleoSpin Soil kit (Macherey-Nagel) starting from 25 mg of material.

3. 16S gene was amplified using 5 µL of the extracted DNA in a final reaction volume of 25 µl using Platinum Taq DNA Polymerase High Fidelity (Thermofisher) in accordance with the manufacturer’s instructions. The amplifications were performed for 26 cycles using 55 °C as the annealing temperature. The following primers were used: Pro341F: 5′-CCTACGGGNBGCASCAG-3′ and Pro805R: Rev 5′-GACTACNVGGGTATCTAATCC-3′.

4. The overhang adapter was added and followed by library construction. The libraries were purified with Beads Amplure XP 0.8X, amplified with Indexes Nextera XT Illumina, and then normalized, mixed, and loaded on Miseq with 2 × 300 bp (paired-end).

Any software that can read fastq files.