Microbial genomic DNA was extracted using DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Mock community DNA was included as positive control for library preparation (Zymobiomics Microbial Community DNA, ZymoResearch, Irvine, CA, USA).
Samples were amplified using primers 341F and 805R, which target the V3–V4 region of the bacterial and archaeal 16S rRNA. PCR was performed in 10 μL final volume with 0.2 μM primer concentration. The PCR included: 3 min at 95 °C (initial denaturation) followed by 25 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, and a final elongation step of 5 min at 72 °C. PCR products were purified using AMPure XP beads (Beckman Coulter, Nyon, Switzerland) with a 0.9× ratio according to the manufacturer’s instructions.
The paired-end sequencing was conducted following an Illumina Miseq sequencing 300 × 2 approach. Quality control filtering and OTU binning of the resulting sequences were executed using DADA2 software [16 (link)]. Finally, taxonomic assignment of phylotypes was performed using a Bayesian classifier trained with Silva database [17 (link)]. Extraction and sequencing of DNA and bioinformatic procedures were carried out by Microomics Systems, S.L. (Barcelona, Spain).
Samples were amplified using primers 341F and 805R, which target the V3–V4 region of the bacterial and archaeal 16S rRNA. PCR was performed in 10 μL final volume with 0.2 μM primer concentration. The PCR included: 3 min at 95 °C (initial denaturation) followed by 25 cycles of 30 s at 95 °C, 30 s at 55 °C and 30 s at 72 °C, and a final elongation step of 5 min at 72 °C. PCR products were purified using AMPure XP beads (Beckman Coulter, Nyon, Switzerland) with a 0.9× ratio according to the manufacturer’s instructions.
The paired-end sequencing was conducted following an Illumina Miseq sequencing 300 × 2 approach. Quality control filtering and OTU binning of the resulting sequences were executed using DADA2 software [16 (link)]. Finally, taxonomic assignment of phylotypes was performed using a Bayesian classifier trained with Silva database [17 (link)]. Extraction and sequencing of DNA and bioinformatic procedures were carried out by Microomics Systems, S.L. (Barcelona, Spain).