2.3. DNA Extraction and DNA Sequence Analysis

The total metataxonomic DNA from a stalactite sample was extracted immediately after collection using a NucleoSpin Soil DNA isolation kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions. Four replications (S1, S2, S3, S4; Supplementary Materials Figures S1–S10) of the same core sample were used for the DNA extraction with different parameters. The DNA quality and concentration were measured by Quawell UV-Vis Spectrophotometer (Q5000). The DNA samples were stored at −20 °C until the library preparation was performed.

Stalactite core samples were sequenced at the Institute of Applied Biosciences. Illumina’s 16S Metataxonomics Protocol (Part # 15044223 Rev. B) was applied for the amplification of V3 and V4 regions of the bacterial 16S rRNA gene using the following primers containing the Illumina overhang adapters. The forward and reverse universal primer sequences, as reported by Klindworth et al. [24], are, respectively, 5′-TCGTCGGCA GCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′ and 5′-GTCTCGTG GGCTCGGAGATGTGTATAAGAGACAGGACTACHV GGG-TATCTAATCC-3′ and create an amplicon of approximately 460 bp. For PCR amplification, 2× Kapa HiFi Hot-Start ReadyMix was used, and the PCR products were then cleaned using Beckman Coulter Agencourt AMPure XP Beads according to the 16S Meta-genomics protocol. The indices and Illumina sequencing adapters were attached using the Nextera^® XT Index Kit, followed by a second clean-up with Beckman Coulter Agencourt AMPure XP Beads. The libraries’ quantification was performed using a Qubit™ 3.0 Fluorometer (Life Technology Ltd., Paisley, UK). Moreover, 2 μL of the final libraries was run on a Fragment Analyzer with the method DNF-473-33-SS NGS Fragment 1–6000 bp in order to check the quality and verify the size of the libraries. After library quantification, normalization, and pooling, the concentrations were adjusted to 125 pM and prepared for loading on the Illumina MiSeq according to Illumina’s 16S Metataxonomics Protocol (Part # 15044223 Rev. B). The libraries’ pool was denatured and loaded on the Illumina MiSeq at 12.5 pM and sequenced paired-end (2 × 300) using a MiSeq^® Reagent Kitv3 (600 cycles) (Illumina, Inc., San Diego, CA, USA).

Furthermore, another important issue that needs to be addressed is that inside the stalactite core, the abundance of the bacteria entrapped is scarce with a lower number of reads (less than 500,000) compared to other biological samples (i.e., gastrointestinal tract with up to 2¹⁹ reads). It is obvious that in this case, the use of metataxonomics (16S DNA) is favorable and performs similarly or even better than other metagenomics (i.e., whole shotgun metagenomic sequencing) (Durazzi et al., 2021) [23].