16S sequence of saliva
DNA was extracted from up to 250 L of saliva via the DNeasy Power Soil Pro Kit (Qiagen, Manchester, UK) according to the manufacturers instructions. Following extraction, the DNA was amplified by PCR using the 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACNVGGGTWTCTAAT-3) primers to amplify the variable region 4 (V4) of the 16S rRNA gene. Extracted DNA was added at a volume of 2.5 L, followed by 5 L of 515F primer, 5 L of 806R primer and 12.5 L of KAPA HiFi HotStart ReadyMix (Roche, London, UK) with a final PCR reaction volume of 25 L. Following a denaturing step at 95C for 3 min, PCR was performed with 25 cycles of 95C for 30 seconds, 55C for 30 seconds and 72C for 30 seconds, followed by a final elongation step of 72C for 5 min. Samples were submitted to Deep Seq Next Generation Sequencing Facility at the University of Nottingham (Nottingham, UK) for sequencing via Illumina MiSeq.