Evaluation of fish biodiversity in estuaries using environmental DNA metabarcoding

Library preparation and sequencing Samples were sent to the Kazusa DNA Research Institute (Chiba, Japan) for paired-end library preparation and next-generation sequencing (MiSeq) as detailed by Miya et al. [24] and S1 Text. A two-step PCR for paired-end library preparation was employed in the MiSeq platform (Illumina, San Diego, CA, USA). For the first-round PCR (1st PCR), a mixture of the MiFish- U and MiFish-E was used. After completion of the 1st PCR, the purified target products (ca. 300 bp) were quantified and diluted. The diluted products were employed as templates for the second-round PCR (2nd PCR) that was carried out with dual-index primers. The blanks were prepared during 1st and 2nd PCR to monitor any contamination. No template was used for both blanks to avoid possible contamination. Data preprocessing and taxonomic assignment Data preprocessing and analysis of MiSeq raw reads were performed with a pipeline (MiFish ver. 2.3) using USEARCH v10.0.240 [26]. The following steps were applied: (1) Forward (R1) and reverse (R2) reads were merged by aligning them with the fastq_mergepairs command. (2) Primer sequences were removed from merged reads using the fastx_truncate command. (3) Reads without primer sequences underwent quality filtering using the fastq_filter command to remove low-quality reads (4) Preprocessed reads were dereplicated using the fastx_uniques command and all singletons, doubletons, and tripletons were removed from subsequent analy- sis as recommended [26]. (5) Dereplicated reads were denoised using the unoise3 command to

generate amplicon sequence variants (ASVs) without any putatively chimeric and erroneous sequences [27]. (6) Finally, ASVs were subjected to taxonomic assignments of species names (molecular operational taxonomic units; MOTUs) using the usearch_global command with Table 1. Environmental conditions of sampling stations. Aka River (17th, July) HT LT L1 L2 R1 R2 Water temp (˚C) 24.1 26 26 26 25.4 25.5 Salinity 6.4 7.7 18.8 29 20.1 29.7 Filtered water (mL) 200 200 600 600 600 600 Tama River (29th, June) HT LT L1 L2 R1 R2 Water temp (˚C) 22.3 26.6 - 24.7 23.5 - Salinity 29.1 22.2 - 24.6 27.9 - Filtered water (mL) 400 400 - 400 400 - Miya River (7th, June) HT LT L1 L2 R1 R2 Water temp (˚C) 23 22.5 22.2 22.6 23.1 24.7 Salinity 7.45 10.4 25.53 23.39 20.64 21.92 Filtered water (mL) 700 600 200 500 600 600 Takatsu River (16th, July) HT LT L1 L2 R1 R2 Water temp (˚C) 26.7 24.1 29 29 27 27 Salinity 0.1 0.1 22.4 23.9 15.1 15.5 Filtered water (mL) 500 600 500 500 500 600 Sendai River (27th, July) HT LT L1 L2 R1 R2 Water temp (˚C) 28.7 30 29.7 29.5 29.1 29.3 Salinity 22.2 6.8 14.2 14.6 28.3 29.6 Filtered water (mL) 500 500 500 600 1000 1000 HT: river mouth at high tide; LT: river mouth at low tide; L1: left 500 m; L2: left 1 km; R1: right 500 m; R2: right 1 km. https://doi.org/10.1371/journal.pone.0231127.t001 PLOS ONE Biodiversity of five estuaries PLOS ONE | https://doi.org/10.1371/journal.pone.0231127 October 6, 2020 4 / 15