Lib l kit

A 500 bp region of the 16S rRNA gene (covering V1–V3) was PCR-amplified from 5 ng of extracted DNA sample using primers comprising universal 16S primers 27F (Frank et al., 2008 (link)) and 519R (Lane et al., 1985 (link)) along with Roche GS-FLX Titanium Series adapter sequences (A & B) for 454 pyrosequencing. The forward primers incorporate 12 base unique barcode sequences (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-NNNNNNNNNNNN-AGAGTTTGATYMTGGCTCAG-3′) to enable pooling of samples in the same sequencing run. The appropriate barcoded A-27F and the B-519R (5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-GWATTACCGCGGCKGCTG-3′) primers were used in PCRs with PHUSION Hi-Fidelity DNA polymerase (Thermo Scientific). For the reaction conditions, there was an initial denaturation step of 30 s at 98°C followed by 25 cycles of 98°C for 10 s, 50°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. PCR amplicons were initially checked using agarose gel electrophoresis and purified using Ampure magnetic beads according to the manufacturer's instructions. Amplicon quantification, QC, pooling, and unidirectional sequencing of the samples was performed using the Lib-L kit and the Roche 454 GS-FLX + Titanium series sequencer by the DNA sequencing facility (Dr. Shilo Dickens), Department of Biochemistry, Cambridge University, Cambridge, UK.

Samples for DNA analysis were kept at −80°C until DNA extraction was performed (in 2012). DNA was extracted from the samples using FastDNA® SPIN Kits for Soil and a FastPrep-24 bead-beading machine (MP Biomedicals, Santa Ana, USA), according to the manufacturer's instructions. Bacterial DNA was amplified using the barcoded primers 338F and 926R according to the protocol described in Torondel et al. (2016 (link)). PCR products were cleaned using the Wizard PCR product purification kit (Promega, Fitchburg, Wisconsin, USA) and were then pyrosequenced at the Wellcome Sanger Institute in 2012 using the Lib-L kit on the 454 GS FLX Titanium System (Roche, Branford, Connecticut, USA). Further details on the protocols used to generate 16S rRNA gene sequence data are as described in Torondel et al. (2016 (link)).

DNA was extracted from the saliva samples by means of the Genelute DNA extraction kit (Sigma-Aldrich), and 16S rRNA genes were amplified by polymerase chain reaction (PCR) with primers 27F (with the YM modification) and 519R.^{22 (link), 23} The primers incorporated a unique barcode and Roche 454 adapters. Polymerase chain reaction amplicons were purified, sized, quantified, and pooled in equimolar proportions. Emulsion PCR and unidirectional sequencing of the libraries were performed using the Lib-L kit and Roche 454 GS-FLX Titanium sequencer.