Insights into the representativeness of biodiversity assessment in large reservoir through eDNA metabarcoding

Sampling stations in Três Irmãos Reservoir After analyzing the data from both pilot sampling and the hydrodynamic modeling (see Results section), 14 new stations distributed along the entire reservoir were sampled in October 2021 to establish a broader baseline, hereafter called stations C1–C4 and C6–C15 (Fig 1A and S1 Table). Sampling stations covered each compartment used to evaluate water renovation ratios across the river. Stations C1, C2, C4, C6, C7, C9, C12, C13, and C14 were located in the main water body. Four stations were placed in areas that comprise the arms with (C3, C8, C10) and without tributaries (C11). One station (C15) was located at the entrance to the Pereira Barreto channel that connects the TI reservoir to the neighboring IS reservoir (Fig 1A). Three replicates (R1, R2 and R3) were collected within each station, all 1 meter above the benthic bottom, resulting in 42 samples at different depths across among the 14 stations (S1 Table). The replicates were collected within approximately 10-minutes intervals. All collection, processing, and storage procedures were the same as those described above for the pilot sampling. DNA extraction and sequencing DNA extraction from the Sterivex filters was performed using the DNeasy1 Blood & Tissue kit (Qiagen) with adaptations [28]. The DNA concentration was determined using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). DNA samples are registered in the Brazilian National System for the Management of Genetic Heritage and Associated Traditional Knowl- edge (SISGEN), accession number A7E42BC.

The amplicons from mitochondrial genes targeting the minibarcodes of cytochrome c oxidase I (COI), 16SrRNA (16S), and 12SrRNA (12S) were amplified and sequenced using the pair of primers described in S2 Table. Amplification was performed in 25 μl using Platinum™ Multiplex PCR Master Mix (Applied Biosystems), 1 μM of each primer, and 5 μl of DNA. The thermocycling conditions included an initial denaturation step at 95º C for 2 minutes and then 25 denaturation cycles at 95˚C for 30 seconds each, annealing at different temperatures for each primer pair (48ºC for CO1; 57ºC for 12S and 53ºC for 16S) for 90 seconds, and extension at 72˚C for 30 seconds, followed by a final extension at 72˚C for 10 minutes and finishing at 4˚C. For the library preparation, the amplicons were indexed using Nextera XT indexes (IDT for Illumina). Library quality control was performed using Tapestation (Agilent). DNA concentration was normalized, and all amplicons were pooled in one library. The library was sequenced on Illumina NovaSeq 6000 S4 Flow Cell 2x150. Bioinformatic analysis After sequencing, raw sequence reads were evaluated using FASTQC (http://www. bioinformatics.babraham.ac.uk/projects/fastqc) and MultiQC (https://multiqc.info/). All gen- erated fastq files were imported into the QIIME2 pipeline [29] for further quality filtering and taxonomic classification. Primer trimming, read denoising, merging the remaining paired reads into amplicons, and chimera removal steps were carried out with DADA2 [30]. Here, all

forward and reverse reads with less than 145 nucleotides overlapping were removed, as well as all reads with quality < 30 in Phred scale. The resulting Amplicon Sequence Variants (ASVs) were clustered into MOTUs (97% of identity among sequences) with DADA2. The taxonomy identification was performed using two different methods: 1) Naive Bayesian classifier [31] trained with the sequences’ dataset of the 12S Mitohelper [32], COI classifier version 4.0 [33] and the MIDORI 16S dataset [34]; and 2) BLASTn searches [35] using the curated PLOS ONE eDNA metabarcoding reservoir PLOS ONE | https://doi.org/10.1371/journal.pone.0314210 January 24, 2025 5 / 22