Plans for collection, laboratory evaluation and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}
Saliva and/or gingival samples collected during each of the follow-ups will be analysed with high-throughput sequencing techniques to characterize the bacteria that are present and to determine bacterial diversity. Bacterial DNA from the 16S rRNA gene will be isolated and the V3–V4 region of the 16S rRNA amplified and sequenced by the Illumina® MiSeq platform. The resulting sequences will be assigned according to taxonomy by the Human Oral Microbiome Database (Quality control and advanced upstream statistical analyses will be performed in QIIME 2 and R. We will model the differential abundance of bacteria in the samples, presented by diversity indices, such as the Shannon index with the QIIME software. The Analysis of composition of microbiomes with bias correction (ANCOM-BC) methodology [