*2.4. Identification of Potentially Biologically Relevant Bacterial Genera by Analyzing Their Frequency of Appearance in the Nasopharynx of Healthy Individuals*

After analyzing taxa abundance at the phylum, family, and genus levels, looking for differences between age and sex groups, we decided to apply another strategy to attempt to identify bacterial genera whose presence in the nasopharynx might be characteristic of a certain life stage, independently on their abundance levels. Thus, based on the idea that in some cases the presence of a genus within a certain sex or age group but not in others can be biologically relevant, even if its abundance is low, we studied the frequency with which each genus appeared in the individuals included in this study by visualizing, for each genus, the percentage of people from each age group in which it is present. Firstly, we analyzed this in the 11 genera that presented significant differences between the distinct age groups, with the aim of checking whether these genera were present in a high percentage of individuals and, therefore, that the relative abundance results previously shown in Figure 4a were reliable. Indeed, our results showed that these 11 genera appeared with a high frequency in the individuals included in this study, especially in the cases of *Acinetobacter*, *Dolosigranulum*, *Haemophilus*, *Moraxella*, *Pseudomonas* and *Staphylococcus* (Figure 4b). These data also revealed that several genera, such as *Brevundimonas*, *Finegoldia* and *Peptoniphilus*, appeared less frequently in the youngest people than in the other age groups, while the frequency of appearance of others, such as *Rothia*, decreases in the oldest people (Figure 4b). Interestingly, it coincides that these genera that appear most frequently in the individuals included in this study are also the ones that appear most often as the dominant genus in the taxonomic composition of individuals (Figures S9 and S10). Next, we identified several bacterial genera that could be biologically relevant as part of the nasopharyngeal microbiota, although they did not show any significant differences in relative abundance between age or sex groups. This was (i) because their frequency of appearance in the nasopharynx of the healthy population was very high, as in the case of *Anaerococcus*, *Burkholderia*, *Campylobacter*, *Delftia*, *Prevotella*, *Neisseria*, *Propionibacterium*, *Streptococcus*, *Ralstonia*, *Sphingomonas* and *Corynebacterium* (Figure 5a); (ii) because their frequency of appearance drastically increased with age, such as the cases of *Faecalibacterium*, *Stenotrophomonas* and *Phascolarctobacterium* (Figure 5b); or (iii) because their frequency of appearance drastically decreased with age, such as the cases of *Aggregatibacter*, *Gemella* and *Fusobacterium* (Figure 5c).

**Figure 5.** Frequency of appearance of potential biologically relevant bacterial genera. (**a**–**c**) Percentage of individuals, of the total included in this study, in which the indicated genera are present in the indicated age groups.
