Metagenomic DNA: floor 2 from the cave 1
Total DNA extractionTotal microbial DNA was extracted from 0.25 g of each sample using the PowerSoil DNA Mobio kit (GeneWorks, Australia), following the protocol recommended by the manufacturer. After extraction, the DNA samples were quantified and their purity was analyzed using NanoDrop  (Thermo Fisher Scientific, USA). The sample quality was verified by electrophoresis on 0.8% agarose gel.Partial amplification of the 16S ribosomal gene for metagenomic analysesFor partial amplification of the 16S ribosomal gene, 515F and 926R oligonucleotides were used to generate an amplicon for the V4-V5 region. These oligonucleotides were designed to include the adapter sequences used in the Ion Torrent sequencing protocol and were linked to a 10 nt barcode for each sample (FOUHY et al., 2016). The complete list of oligonucleotides used in this study can be found in Supplementary Table 1. The PCR was prepared in a final volume of 30 l containing 0.38 mM dNTP, 2.5 mM MgCl 2 , 0.15 M of each oligonucleotide, 20 ng DNA, 0.08 U of Platinum Taq DNA polymerase (Invitrogen), and 10x Buffer. Amplification was performed in a thermocycler (Biocycler) with an initial cycle of 5 min at 94C, followed by 35 cycles of 30 s at 94C, 1 min at 57C, and 1 min at 72C, with a final cycle of 5 min at 72C. The amplicons were analyzed on an 0.8% agarose gel. Once quality was checked, 22 l of the final PCR product was purified using the GFX PCR Kit and Gel Band Purification (GE Healthcare) Kit, following manufacturer&#39;s recommendations. The quality of the purified product was again measured on 0.8% agarose gel.Metagenomic sequencing using the Ion Torrent platformThe DNA library was quantified using Qubit dsDNA HS (High Sensitivity) using the Qubit 2.0  fluorometer (Life technologies, USA) and the samples were then normalized to 70 pM. For library assembly, 20 l of each sample was placed in a single 1.5 mL tube and homogenized. Then, 25 l of this DNA pool was loaded onto a 318 v2 chip (Life technologies, USA), and the samples were sequenced using the Ion PGM platform (Life technologies, USA) following the manufacturer&#39;s recommendations. After sequencing, the low quality reads and polyclonal sequences were filtered using the software of the equipment itself and the data obtained were exported as FastQ files.