4.3. Stool Sampling and 16S rRNA Gene Sequencing

Stool samples (1 g) were collected 96–120 h after birth (day 4) from a disposable diaper using a sterilized spoon provided with the container, and stored at −80 °C within 2 h of sampling. The samples were thawed, and DNA was extracted using a NucleoSpin DNA Stool Kit (MACHEREY-NAGEL, Düren, Germany). Hypervariable regions of the DNA, and v2, v3, v4, v6-7, v8, and v9 of the 16S rRNA region, were multiplex amplified using a 16S metagenomics kit, following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). A library was generated after refinement using the Ion Plus Fragment Library Kit and Ion Xpress Barcode Adapters Kit (Thermo Fisher Scientific). The Ion Universal Library Quantification Kit with the Quant Studio 5 system (Thermo Fisher Scientific) was used to quantify and pool the barcoded library in order to generate a final concentration of 50 pM per target. The Ion Chef Instrument and the corresponding kit were used to achieve target concentrations for template preparation and emulsion PCR. Sequence analysis was performed using the Ion Gene Studio S5 System and Ion 530 chip (Thermo Fisher Scientific). The sequences were then clustered into OTUs with 97% identity using the MicroSEQ 16S Reference Library v2013.1 and Greengenes v13.5 databases. The raw BAM file was analyzed using Ion Reporter Software with the Metagenomics 16S w1.1 v5.16 workflow (Thermo Fisher Scientific), with default settings (read length filter: 150; minimum alignment coverage: 90.0; read abundance filter: 10; slush ID reporting percentage: 0.2). Ion Reporter leverages Quantitative Insights Into Microbial Ecology (QIIME)’s open-source bioinformatics pipeline, which was used to produce alpha and beta diversity analyses and visualizations.