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.