title: Bacteria 16S rRNA gene V4 variable region in the saliva of homo sapiens: saliva
design: Unstimulated saliva was collected in humans after rinsing the mouth. Collected saliva was centrifuged at 6,000  g at 4 for 30 min. Total microbial DNA was extracted from saliva samples using a QIAamp DNA Microbiome Kit (QIAGEN) according to the manufacturers standard protocol. DNA extracts were quantified using the Quant-iT PicoGreen kit (Invitrogen, ThermoFisher Scientific). 16S rRNA gene amplicons covering variable regions V3 to V4 were generated using primers (515F and 806R with Illumina adapters and 8 basepair dual indices). The PCR reaction was incubated at 95C for 2 min, the 30 cycles of 30 s at 95.0C, 60 s at 50.0C, and 60 s at 72.0C, followed by a final extension at 72.0C for 10 min. PCR products were pooled for quantification and purified using the Qiaquick PCR purification kit (QIAGEN Inc.) following the manufacturers specifications. 16S libraries were sequenced on the MiSeq using a v2 2x250 base-pair kit (Illumina, Inc)