title: DNA_16sH18
design: Next generation sequencing was used to study the biodiversity of the Lake Enigma study sites. The V3-V4 variable regions of SSU rRNA gene were amplified using following primer pair, S-D-Bact-0341-b-S-17 (5 -CCTACGGGNGGCWGCAG-3) and S-D-Bact-0785-a-A-21 (5-GACTACHVGGGTATCTAATCC-3) (Herlemann et al., 2011). Amplicons were sequenced on the Illumina MiSeq platform by FISABIO, Valencia, Spain (http://fisabio.san.gva.es/en/servicios). Library preparation and Illumina sequencing were performed according to standard protocols (Caporaso et al., 2012). Raw reads were cleaned of barcodes and primers, then reads shorter then <150 bp or with ambiguous base calls, and with homopolymer runs exceeding 6 bp, were removed. Reads were imported in RStudio package (R Core Team, 2022) and analyzed using the DADA2 package (v 1.26.0; Callahan et al., 2016) as described in (Cordone et al., 2021). Due the preprocessing steps done by sequencer facility (FISABIO, Valencia, Spain) the reads were trimmed using modified filtering parameters (truncLen=c(280,280), maxN = 0, truncQ = 0, rm.phix = TRUE) according to (Crisafi et al., 2022). A parametric error model, based on the convergence between the estimation of error rate and the inference of the sample composition, was applied to remove PCR biases (Callahan et al., 2016). Then paired-end reads were merged and the amplicon sequence variants (ASVs) table was constructed. Chimeric sequences were removed, and the taxonomy was assigned to sequence variants using the naive Bayesian classifier method against the Silva Database v138 (v138; https://www.arb-silva.de/documentation/release-138/, and https://zenodo. org/record/4587955#.YgKJlb_MJH4).