2.3. Microbiota determination

Samples were slowly defrosted at −20 °C for 24 h and 4 °C for another 24 h, to avoid bacterial death and DNA fragmentation. Total DNA was obtained by the QiaAmp kit (QIAGEN) from the saliva pellet after centrifugation. The bacterial composition was determined by polymerase chain reaction (PCR) amplification of the 16S rDNA V3–V4 region, and PCR products were pooled equally and submitted to massive sequencing (2x300 bp) on a MiSeq (Illumina, San Diego, CA, USA) platform at traslational genomics core support unit (UCAT) -Ramon y Cajal Health Research Institute (Madrid, Spain). The sequence's quality control was performed with DADA2, with a rarity of 12,000 sequences per sample. Amplicon sequencing variants were obtained by taxonomic assignment with the Silva_132 classifier. Alpha and beta diversity studies were performed, employing the q2-diversity add-on of QIIME2, after normalizing the samples by rarefaction (subsampling without replacement). In addition, a linear effect size discriminant analysis (LEfSe) was performed to assess which taxa explained the differences between groups. Sequence data were deposited in Genbank (BioProjectPRJNA1054537).