title: DNA-16s-water
design: Biodiversity of microorganisms colonizing the masks was investigated by means of Next Generation Sequencing with universal primers targeting The V3-V4 hypervariable regions of the prokaryotic SSU rRNA gene (Forward = 5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG Reverse = 5' GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC, Klindworth et al., 2013) were PCR amplified and the amplicons were sequenced on the Illumina MiSeq platform by a commercial company (FISABIO, Valencia, Spain) (http://fisabio.san.gva.es/en/servicios). Libraries preparation followed by Illumina sequencing were performed according to standard protocols (Caporaso et al., 2012). Sequences were deprived of barcodes and primers, then sequences <150bp together with sequences with ambiguous base calls and with homopolymer runs exceeding 6bp were removed. All sequences were imported in R, and analyzed with the DADA2 package (Callahan et al., 2016) as described in (Cordone et al., 2021). Due to the preprocessing steps done by sequencer facility (FISABIO, Valencia, Spain) the reads were trimmed using the filterAndTrim command using these settings (truncLen=c(280,280), maxN=0, truncQ=0, rm.phix=TRUE). After this step, a parametric error model, based on the convergence between the estimation of error rate and the inference of the sample composition, was performed. Paired-end reads were merged and exact Amplicon Sequence Variants (ASVs) inferred using the dada algorithm. Chimeric sequences were removed and prokaryotic taxonomy was assigned using the naive Bayesian classifier method against the Silva Database (v138; https://www.arb-silva.de/documentation/release-138/, and https://zenodo.org/record/4587955#.YgKJlb_MJH4). ASVs abundance table obtained from DADA2 was further processed in R using Phyloseq, Vegan, and Microbiome packages (McMurdie and Holmes, 2013; Oksanen et al., 2020; Lahti and Shetty, 2017). uses evolutionary modeling and a reference genome database (Ahauer et al., 2015), default values were applied.