Refining sampling efforts for fish diversity assessment in subtropical urban estuarine and oceanic waters using environmental DNA with multiple primers

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 differe..., 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)..., , # 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 (#...