Higher abundance of Actinomyces spp. associated with spontaneous preterm birth in overweight mothers

Microbial analysisDNA extraction and PCR amplificationMicrobial DNA were extracted from the stool samples using the QIAmp DNA Stool mini kit (Qiagen, Germany), according to the manufacturer's protocol. The V3–V4 region of the bacterial 16S rDNA gene was amplified by PCR (95°C for 3 min, followed by 30 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, and a final extension at 72°C for 10 min) using primers F 5’-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3’ and R 5’-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3’ [29]. The amplicon DNA predicted size was about 550 bp and be verified by FragmentAnzlyzer (Agilent Technologies Inc.). Amplicon DNA, therefore, would be purified by KAPA Pure Beads (Roche) according to manufacturer’s protocol. Library index PCR would perform by Illumina Nextera XT DNA Library Preparation Kit Set A.Illumina MiSeq sequencingAmplicons were purified using the KAPA Pure Beads DNA (Roche) according to the manufacturer's instructions and quantified using Qubit 4 fluorometer (Thermo Fisher Scientific). Purified amplicons were pooled in 10 pM and paired-end sequences (2×300) on an Illumina MiSeq platform (MiSeq v3 600 cycle kit) according to the standard protocol.Bioinformatic analysis More than 100,000 sequencing reads were picked from each sample using a pseudorandom generator for comparison of community composition and structure among samples because the lowest sequencing read of all the samples was >100,000. The saturation of α-diversity was performed by Shannon Diversity (including richness and evenness). Raw Fastq files were demultiplexed and quality-filtered using QIIME 2 (version 2021.4) and denoising by DaDa2. Operational taxonomic units (OTUs) were clustered with a 97% similarity. The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the Greengene (v13.8) 16S rDNA database using confidence threshold of 70%.