DOI: 10.5061/dryad.08kprr58c
title: Data from: Refining sampling efforts for fish diversity assessment in subtropical urban estuarine and oceanic waters using environmental DNA with multiple primers

description: The environmental DNA (eDNA) approach is an emerging tool for monitoring
 marine biodiversity. However, the sampling effort needs optimization
 according to the site characteristics and target taxonomic groups. In this
 study, we optimized the eDNA sampling effort in terms of sample volume and
 number of replicates to monitor the diversity of marine vertebrates
 (mainly fish) in Hong Kong's subtropical waters that show a gradient
 of estuarine to oceanic waters. To maximize detection, we used three pairs
 of metabarcoding primers (12S‐v5, MiFish‐U, and MiFish‐E). We compared
 vertebrate diversity in 78 water samples, ranging from 1 to 10 L,
 collected from oceanic and estuarine sites. Metabarcoding yielded a total
 of 140 vertebrate species, of which 18 were unique to the estuarine site,
 66 unique to the oceanic site, and 56 shared between both sites. The
 detected species were predominantly ray‐finned fish (136 species), and the
 three primer pairs exhibited differential sensitivity toward different
 taxa, especially cartilaginous fish and cetaceans. Increasing sampling
 volume per replicate generally increased the total detected species,
 average species per replicate, and species coverage, and sampling 3 or 4 ×
 4 L represented the most efficient sampling effort for the estuarine and
 oceanic sites, respectively. The diversity analysis revealed that sampling
 &gt;2 L per replicate reduced variability and improved diversity
 analysis. The results also showed that a larger sampling volume per
 replicate increased the probability of detecting endangered, indicator,
 invasive, and elusive species, with 4 L representing the most efficient
 volume. This study recommended sampling 4 L per replicate and 3 replicates
 for estuarine and 4 for oceanic sites, respectively for effectively
 monitoring marine fish in subtropical waters using the eDNA approach.
description: Marine water samples were collected 1 m beneath the water surface
 using an electric water pump and randomly distributed into sterile
 sampling bags of different volumes. The samples were collected from an
 estuarine site (22.39423, 113.90088) on 24 March 2023 and an oceanic site
 (22.351714, 114.350139) on 15 July 2023. The water samples were filtered
 using 0.45 micron pore size glass fiber membranes, and the eDNA was
 extracted using CTAB-chloroform methods. The target genes were amplified
 using 12S-v5 (Riaz), MiFish-U, and Mifish-E primers. PCR reaction
 solutions containing KOD polymerase premix (Toyobo, Osaka, Japan), 200 nM
 primers, and 1.0 ng/μL DNA template were subjected to a 5-min initial
 denaturation stage at 98 °C, followed by 35 cycles of 5 s 98 °C, 5 s 60
 °C, and 10 s 68 °C, and a 5 min final extension stage at 68 °C. The field
 control was also included as a negative control. The successful reactions
 were purified from the gel using a gel extraction kit (Qiagen, Hilden,
 Germany), adjusted to the same concentration, and pooled (10 samples per
 run) before being sent to a commercial sequencing service provider
 (Novogene Co. Ltd., Beijing, China) for paired-end 150 bp library
 preparation and sequencing on a Novaseq 6000 platform (Illumina, San
 Diego, CA, USA). The sequences were quality-filtered, demultiplexed
 (sabre), removed of primer sequences (cutadapt), and denoise &
 merged (Qiime2-dada2).
description: # Raw sequences from estuarine and oceanic sites
 [https://doi.org/10.5061/dryad.08kprr58c](https://doi.org/10.5061/dryad.08kprr58c) These are the raw sequences produced from an eDNA study in estuarine and oceanic sites in Hong Kong. ## Description of the data and file structure This dataset includes raw sequencing data of three primer pairs: 12S-V5, MiFish-U, and MiFish-E. The pair-end raw sequences were demultiplexed into 80*2 fastq files for every primer, with XXX_1.fq as forward reads, XXX_2.fq as reverse reads. However, due to the use of PCR-free library preparations, the reads were mixed-oriented. The reads could be re-oriented using *cutadapt*, by adding *--revcomp* command during primer removal steps. We studied two sites in this study: "estuarine" and "oceanic". The filenames starting with "west" represent the estuarine samples, whereas those with "east" represent the oceanic samples. The samples are further divided into eDNA extracted from 1 L(#01-20), 2 L (#21-30), 4 L (#31-35), and 10 L (#36-39) seawater, whereas #40 represents negative control for each site. The naming is the same for three datasets: 1-12S (12S-V5) 2-MiFish-U 3-MiFish-E