16S rRNA sequencing of saliva
The variable regions V1-V2 of the 16S rRNA gene were amplified by polymerase chain reaction (PCR) using a previously published method 27 using 27F (with YM modifications) and 338R primers with Illumina (Illumina, Cambridge, UK) barcodes and adapters, amplification products were purified using the QiaQuick PCR Purification kit (Qiagen, Manchester, UK), and libraries were pooled for sequencing at a concentration of 10 nM. Samples were sequenced at the Queen Mary University of London Genome Centre using the Illumina MiSeq (Illumina) platform v3 2 _ 250 bp flow cell for paired-end sequencing.  Sequences were obtained in fastq format and were analysed using the dada2 pipeline 28 (version 1.21.0) in RStudio (version 1.4.1717) and sequences were assigned genus/species using the Human Oral Microbiome Database (HOMD; version 15.9) 16S rRNA database, and alpha and beta diversity was calculated using RStudio. Packages used were phyloseq, ggplot2, readr, plyr, reshape2, RColorBrewer, ggpubr, vegan, ANCOMB and Boruta.