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DNA extraction, amplification, and sequencing of the 16S rRNA gene
Total DNA is extracted from samples of different matrices (fecal, urinary, oral, and vaginal) using a commercial kit from QIAGEN (Hilden, Germany, DNeasy Blood & Tissue Kit) with an initial modification to the protocol. The material is added to four 3 mm glass beads and 0.5 g of 0.1 mm zirconia beads (BioSpec Products, Bartlesville, OK, USA), followed by three homogenization steps using the FastPrep instrument (MP Biomedicals, Irvine, CA, USA) at 5.5 m/s for 1 minute. Finally, the samples are incubated at 95 °C for 15 minutes to perform both mechanical disruption and thermal shock. The extracted DNA is then stored at –20 °C for subsequent analyses. DNA quantification is performed using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, DEU), which allows for its subsequent dilution to the working concentration.
The subsequent preparation of sequencing libraries is performed following the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Illumina, San Diego, CA, USA). Specifically, the V3–V4 hypervariable regions of the 16S rRNA gene are amplified through a PCR reaction in a final volume of 50 μL containing genomic DNA (25 ng), 2X KAPA HiFi HotStart ReadyMix (Roche, Basel, Switzerland), and 200 nmol/L of primers 341F and 785R, carrying the Illumina adapter sequences.
The PCR thermal cycle consists of an initial denaturation step (95 °C for 3 min), followed by 25 cycles of denaturation (95 °C for 30 s), primer annealing (55 °C for 30 s), and DNA extension (72 °C for 30 s). The reaction concludes with a final extension at 72 °C for 5 min.
The resulting PCR products are purified using Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). The indexed libraries are then prepared through a limited-cycle PCR using Nextera technology and purified again as above using magnetic beads. The libraries are subsequently quantified using a Qubit 3.0 fluorometer (Invitrogen, Waltham, Massachusetts, USA), normalized to a concentration of 4 nM, and pooled into a single Eppendorf tube. The pooled libraries are then denatured with 0.2 N NaOH and diluted to a final concentration of 5.5 pM with a 20% PhiX control.
The final sequencing is then performed on an Illumina MiSeq platform using a paired-end 2 × 250 bp or 2 × 300 bp protocol, following the manufacturer’s instructions (Illumina, San Diego, CA, USA).
Metabarcoding: bioinformatics and biostatistics
The raw sequences obtained from the sequencer are analyzed using a pipeline that combines PANDAseq (Masella et al., 2012) and QIIME 2 (Bolyen et al., 2019). High-quality reads (minimum/maximum length = 350/550 bp) are obtained using the fastq filter function of the Usearch11 algorithm (Edgar, 2010) and subsequently grouped into ASVs (Amplicon Sequence Variants) using DADA2 (Callahan et al., 2016). Taxonomic classification is performed using the VSEARCH algorithm (Rognes et al., 2016) and the SILVA database (Quast et al., 2013). All unassigned sequences are discarded.
Alpha diversity is calculated using the Shannon index metric, and the analyses and graph production are performed in R and Python.
Shotgun metagenomic sequencing
The extracted and frozen DNA obtained through the extraction methods described above is also used for shotgun metagenomic sequencing, which allows for massive sequencing of the entire metagenome of the sample. DNA library preparation for shotgun sequencing is performed using the QIAseq FX DNA library kit (QIAGEN) according to the manufacturer’s instructions.
Specifically, 100 ng of total DNA for each sample are fragmented into 450 bp DNA fragments, with end-repair and A-tailing, using the FX enzymatic mix (included in the kit) and the following thermal cycle: 4 °C for 1 min, 32 °C for 8 min, and 65 °C for 30 min. The libraries are then incubated at 20 °C for 15 min for the ligation of Illumina adapters in the presence of DNA ligase, followed by purification of the products using Agencourt AMPure XP beads, a subsequent 10-cycle PCR amplification, and an additional purification with magnetic beads. The samples are then mixed in equimolar concentrations (4 nM) to obtain the final library. Sequencing is then performed on an Illumina NextSeq platform using a paired-end 2 × 150 bp protocol, following the manufacturer’s instructions.
Metagenomics: bioinformatics and biostatistics
The raw reads obtained from the sequencer are processed to remove reads originating from the host’s eukaryotic DNA using KneadData, with the host genome retrieved from the reference deposited in NCBI GenBank. After filtering, the reads are processed with fastp to remove short and low-quality reads and to eliminate duplicates. The resulting high-quality reads are then used for taxonomic assignment through k-mer–based approaches (Kraken2 and the Kraken2 Standard reference database) and for functional assignment using pipelines that enable accurate profiling of the presence/absence and abundance of bacterial pathways within microbial communities (HUMAnN).