Miseq fgx platform

Total genomic DNA was extracted from salivary samples using the DNeasy PowerSoil kit by following the manufacturer’s instructions. Sequencing libraries were prepared by following the Illumina 16S Metagenomic Sequencing Library Preparation workflow. The V3-V4 regions of the 16S rRNA gene were amplified with the bacterial universal primer combination (341F-5′-CCTACGGGNGGCWGCAG-3′ and 806R-5′- GGACTACHVGGGTWTCTAAT-3′), which contained a barcode sequence for Illumina MiSeq sequencing from each sample (55 (link)). The PCR products were pooled and cleaned using the Qiagen Gel Extraction kit (Qiagen, Hilden, Germany) and were then sequenced with the Illumina MiSeq FGX platform. Negative controls consisting of sterile water were included during DNA extraction and sequencing.
The raw 16S rRNA gene amplicon sequences were merged, quality filtered and clustered by using a combination of QIIME (56 (link)), VSEARCH (57 (link)) and USEARCH (58 (link)). Chimeric sequences were removed based on the UCHIME algorithm (59 (link)). The sequences were clustered into operational taxonomic units (OTUs) with 97% similarity. Following these steps, the OTU sequences were obtained. The taxonomic assignment of the representative OTU sequences was annotated by matching the Human Oral Microbiome Database (eHOMD 16S rRNA RefSeq Version 15.22) (60 ).

The PCR products were purified with the QIAquick 96 PCR Purification Kit (Qiagen) and the TruSeq DNA PCR-Free HT Kit (Illumina, San Diego, CA, USA) and used for library preparation, according to the manufacturer’s guidelines. The libraries were sequenced on a MiSeq FGx platform (Illumina) using the Miseq Reagent Kit v2 (Illumina), with a read length of 250 bases.