16S rRNA sequencing of Homo sapiens: vaginal swabs from pregnant women at high risk of preterm birth
Total DNA was extracted from vaginal and rectal swabs using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research Europe GmbH) with modified bead-beating for enhanced mechanical lysis. DNA quality and quantity were assessed using Qubit fluorometry. The V3V4 hypervariable regions of the 16S rRNA gene were PCR-amplified using primers 341F (CCTACGGGNGGCWGCAG) and 785R (GACTACHVGGGTATCTAAKCC), as described by Klindworth et al. Amplicons were purified and indexed according to Illumina's standard protocols. Sequencing was performed on an Illumina MiSeq platform using 2  300 bp paired-end reads by LGC Genomics (Berlin, Germany). Samples were stored at 80 C prior to extraction and sequencing.