Microbiome Analysis of Environmental Samples

After 6 months of exposure, from 3 replicates per material, 5 circular pieces of 0.3 cm² were cut with a hole puncher and placed in cryotubes containing DNA/RNA Shield stabilization solution (Zymo Research Corporation, Tustin, CA, USA). Samples were sent to Microomics Systems S.L. (Barcelona, Spain) where they were frozen at −80 °C until analyzed. DNA was extracted using the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The extraction tubes were agitated using Tissue lyser II (Qiagen, Hilden, Germany) at 30 Hz/s for 10 min. Mock community DNA was included as positive control for library preparation (Zymobiomics Microbial Community DNA, ZymoResearch, Irvine, CA, USA) and to ensure quality control. Samples were amplified using 16S rRNA V3-V4 regions specific primers (V3-V4-Forward 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′, V3-V4 Reverse 5′GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′). Amplicon library preparation was performed as previously reported [20 (link)]. Negative controls included sample collection buffer, DNA extraction and PCR amplification blank. Sequencing was performed using an Illumina MiSeq (2 × 300 bp).