NGS of PCE dechlorinating consortium
4 ng of DNA extracted from each consortium were used for NGS analysis. DNA extraction 16S rRNA Amplicon Library Preparation targeting the V13 variable region was constructed (Matturro et al., 2020). In detail, PCR reactions were performed in 25 L total volume containing Phusion Master Mix High Fidelity (Thermo Fisher Scientific, United States) and 0.5 M final concentration of the library adaptors with V1V3 primers (Bacteria, 27F: 5-AGAGTTTGATCCTGGCTCAG-3; 534R: 5-ATTACCGCGGCTGCTGG-3). Libraries were purified using the Agencourt AMpureXP-beads protocol (Beckmann Coulter, Italy) and the concentration was measured with Qubit 3.0 fluorometer (Thermo Fisher Scientific, Italy). Purified libraries were pooled in equimolar concentrations and diluted to 4 nM. Phix control (15%) was added at 10% in the pooled libraries to overcome issues often observed in 16S rRNA sequencing. Samples were paired-end sequenced (2  301 bp) on a MiSeq (Illumina, United States) instrument using a MiSeq Reagent kit v3, 600 cycles (Illumina, United States) following the standard guidelines. NGS raw data were processed and analyzed using QIIME2 software tools 2018.2 release. The reads were demultiplexed using demux plugin1, denoized, dereplicated and chimera-filtered using DADA2 algorithm (Callahan et al., 2016). The taxonomic analysis was based on a NaveBayes classifier trained on 16S rRNA gene sequences clustered at 99% similarities within the Silva 132-99 database (release December 2017, https://www.arb-silva.de/documentation/release-132/), allowing to construct a data set of amplicon sequence variants (ASVs).