Saliva sample collection, 16S rRNA sequencing and processing
A detailed description of saliva sample collection, 16S rRNA isolation, sequencing, and read processing was reported previously.20 In summary, a total of 143 saliva samples were collected at the inclusion time in falcon tubes and stored at -80°C in each center until the shipment to a long-term storage biobank in Regensburg, Germany. Polymerase chain reaction (PCR) amplification using specific primers (hypervariable V3-V4 selective of 16S rRNA) was performed. Based on standard Illumina protocol, samples were sequenced by MiSeq V3 2\(\times\)300 bp. Sequencing of more than 10,000 reads per sample was considered. After the quality control using MultiQC,21 and FastQC, 22 primers were removed by Cutadapt, 23 and then the DADA2 pipeline,24 was followed (GitHub: https://benjjneb.github.io/dada2/tutorial.html). Furthermore, according to the SILVA database v138, 25 amplicon sequence variants (ASVs) were annotated to their respective bacterial taxonomy.