Dried Saliva Stains

With the exception of the fresh (t0) saliva swabs that were processed straightaway, the prepared swabs were dried and directly exposed to our laboratory’s environment apart from each other, for a specific time prior to bacterial DNA isolation. The swabs were stored at standard room temperature (20–25°C), with relative humidity (30-50%), and daily ambient light (8-11 h). Ambient light included both artificial and natural light sources as the swabs were placed 4 m away from a window (though not directly hit by the sun). First, for the long-term time-dependent bacterial composition analysis, saliva was collected from two individuals (Figure 1B). To sufficiently cover selected time points over a 1-year period, seven saliva swabs were prepared per individual (single sample replicates per time point) and processed at day 1 (t1), 7 (t2), 30 (t3), 120 (t4), 180 (t5), and 365 (t6) after deposition. Second, for the short-term time-dependent bacterial marker analysis, saliva was collected from 15 individuals (Figure 1C). To sufficiently cover selected time points over a 1-month period, 32 saliva swabs were prepared per individual (double replicates per time point); with the exception of one individual (No 1) for which there was insufficient volume of saliva to prepare the last time point. In this case, dried saliva swabs were processed at day 2 (t1), 4 (t2), 6 (t3), 8 (t4), 10 (t5), 12 (t6), 14 (t7), 16 (t8), 18 (t9), 20 (t10), 22 (t11), 24 (t12), 26 (t13), 28 (t14), and 30 (t15) after deposition. Additionally, six of these fifteen individuals also donated saliva 7 months after the first collection date (Figure 1D). For this, eight saliva swabs were prepared per individual (single replicates per time point) and processed at day 2 (t1), 6 (t2), 10 (t3), 14 (t4), 18 (t5), 22 (t6), 26 (t7), 28 (t8 for individual No 1), and 30 (t8 for the rest of individuals) after deposition, corresponding to 1-month time frame as in the first collection date. Additionally, swabs with no biological material were prepared as background blanks in both the long-term and short-term experiments and were exposed and processed in parallel at the same time points as the dried saliva stains.