**4. Methods and Materials**

#### *4.1. Sample Selection, Collection, and Classification*

Due to the available economic resources, we randomly selected 120 nasopharyngeal samples from a cohort of 6354 healthy subjects belonging to the Health Area I of the Region of Murcia (Spain) who voluntarily provided their samples between 27 August 2020 and 8 September 2020 for diagnostic purposes and tested negative for SARS-CoV-2 infection. Nasopharyngeal swabs were obtained by approaching the nasopharynx transnasally and stored in Universal Transport Medium (UTM): Viral Transport medium (COPAN Diagnostics Inc., Murrieta, CA, USA). Nucleic acid extraction was performed using the automatized system Nuclisens EasymaG (bioMérieux, Madrid, Spain) based on the ability of silica to bind DNA and RNA in high salt concentrations (Boom technology). The polymerase chain reaction (PCR) kit used to verify that all the samples were negative for SARS-CoV-2 infection was Novel Coronavirus (2019-nCoV) Real Time Multiplex RT-PCR kit (Detection for 3 Genes), manufactured by Shanghai ZJ Bio-Tech Co., Ltd. (Liferiver Biotech, la Jolla, CA, USA) and the CFX96 Touch Real-Time PCR Detection System (BioRad, Madrid, Spain).

To facilitate the study of age- and sex-associated changes in the nasopharyngeal microbiota throughout life, and to ensure that the sample size of all the age and sex groups were homogeneous, we decided on an experimental design that distributed the 120 nasopharyngeal samples that we could analyse into six age groups with 20 individuals each, of which 10 were females and the other 10 were males (Table S1). For this, the 6354 healthy subjects of our parent cohort were divided into their age matched groups and numbered, and then randomly obtained numbers were used to select 10 females and 10 males from each of the age groups. Random numbers were generated in RANDOM.ORG, which is a True Random Number Generator (TRNG) that generates true randomness via atmospheric noise, unlike the most common and less trustworthy Pseudo-Random

Number Generators (PRNGs) [RANDOM.ORG: True Random Number Service. Available at: https://www.random.org]. According to the exclusion criteria we established for this study, (1) individuals younger than 1 year of age were disqualified because the microbiome of infants is known to be highly fluctuating with age, and (2) subjects who were tested for SARS-CoV-2 infection because they had respiratory or any other kind of symptoms were also excluded to avoid the enrolment of individuals who could have any infection or disease that could alter their nasopharyngeal microbiota although they were not infected by SARS-CoV-2.
