DOI: 10.5061/dryad.ttdz08m73
title: Data from: MiFish data from river and sea water concentrated using glass fiber filters, Sterivex, and QuickConc

description: Environmental DNA (eDNA) analysis is effective for non-invasive
 biodiversity monitoring, revealing species distribution and abundance
 without ecosystem disruption. Concentration, extraction, and preservation
 are three essential steps in the eDNA analysis process. Among these, the
 concentration of eDNA has gained significant research interest,
 particularly due to the variability of water samples used in studies. To
 date, various methods for eDNA concentration have been developed,
 including glass fiber filtration, Sterivex filters, and passive samplers.
 However, no single method is universally applicable because of the
 variability in eDNA presence and water characteristics, such as turbidity.
 Therefore, the development of alternative eDNA concentration methods is
 crucial for advancing eDNA research. This research introduces QuickConc, a
 novel nucleic acid capture method that combines benzalkonium chloride
 (BAC) with dispersed glass fibers. Our results indicate that this approach
 enhances eDNA capture sensitivity by likely improving the interaction
 between silica and eDNA. QuickConc was tested in three environments, using
 metabarcoding and qPCR. Species-specific qPCR results showed that
 QuickConc detected 2 to 3 times higher copy numbers compared to glass
 fiber filter and Sterivex methods. Metabarcoding analyses using the MiFish
 method revealed that the number of fish species detected in river water
 was higher with QuickConc, compared to other methods, while in sea water,
 the number of fish species was at a similar level compared to other
 methods. QuickConc offers new options in eDNA analysis, providing a more
 sensitive and easily deployable approach to biodiversity monitoring and
 conservation strategies.
description: To evaluate the effect of different concentration methods on the
 number of detected species and community composition, metabarcoding
 analyses were conducted for fish eDNA. Our sampling sites included river
 and sea, but notably excluded pond samples from the sequencing process.
 The decision to omit pond samples was grounded in the site’s unique
 characteristics; originally a rice field situated in a segregated area,
 the pond exhibited a limited presence of fish species.
 After DNA extraction, subsequent procedures of amplicon library
 preparation and NGS were performed by Bioengineering Lab. Co., Ltd
 (Kanagawa, Japan). The DNeasy PowerClean Pro Cleanup Kit (Qiagen) was used
 to remove PCR-inhibitory substances, and the elution of DNA was performed
 in a final volume of 50 μL. The library construction began with a two-step
 tailed PCR method using four primer sets (Table S3 in related
 publication), which were partially modified fish universal PCR primers,
 “MiFish,” developed by Miya et al. (2015) to amplify eDNA from fish
 species. The first-round PCR reaction was conducted in
 a 10 μL mixture containing 0.375 U ExTaq HS (TaKaRa), 1× ExTaq Buffer, 0.2
 mM of each dNTPs, 0.5 μM of each primer, 1 μL of eDNA template, and
 ultrapure water. The thermal cycle profile was 94 °C for 2 min, followed
 by 35 cycles of 20 s at 94 °C, 15 s at 65 °C, and 20 s at 72 °C, and a
 final extension of 5 min at 72 °C. After purification of the first PCR
 products using VAHTS DNA Clean Beads (Vazyme), the second-round PCR had a
 10 μL total reaction volume, including 0.2 U KOD FX Neo (TOYOBO), 1 × PCR
 Buffer, 0.4 mM of each dNTPs, 0.25 μM each primer, 2 ng of the first PCR
 product, and ultrapure water. The second PCR’s thermal
 cycles profile consisted of 94 °C for 2 min, 12 cycles at 98 °C for 10 s,
 60 °C for 30 s, and 68 °C for 30 s, with a final extension at 68 °C for 2
 min. PCR products were purified using VAHTS DNA Clean Beads (Vazyme), and
 then the paired-end sequence libraries were completed. The quality of the
 constructed library was verified using a fragment analyzer and the dsDNA
 915 Reagent Kit (Agilent Technologies), after which sequencing was
 conducted on the MiSeq system using the MiSeq Reagent Kit v3 (Illumina)
 under conditions of 2 × 300 bp. Only read sequences
 that were fully matched with primer sequences at the beginning were
 extracted using the fastq barcode splitter in the FASTX Toolkit (ver.
 0.0.14). FASTX-Toolkit's fastx_trimmer was used to remove the primer
 sequences and the last 120 bases at the 3’ end from the extracted reads.
 Subsequently, sequences with a quality score below 20 were filtered out
 using Sickle (ver. 1.33), and any sequences shorter than 40 bases, along
 with their paired reads, were discarded. Following this, the DADA2 plugin
 in Qiime2 (ver. 2024.2) was used to remove chimeric and noisy sequences,
 after which representative sequences and an ASV table were generated. For
 MiFish analyses, the obtained ASV table was analyzed with USEARCH v
 10.0.240 (Edgar 2010), according to the method demonstrated by Wu et al.
 (2021). The mitochondrial genome database MitoFish (ver. 3.90) was used to
 assign taxonomic names to ASVs.
description: # Data from: MiFish data from river and sea water concentrated using glass
 fiber filters, Sterivex, and QuickConc
 [https://doi.org/10.5061/dryad.ttdz08m73](https://doi.org/10.5061/dryad.ttdz08m73) ## Description of the data and file structure This dataset contains metabarcoding data derived from water samples collected across two aquatic environments in Japan: Kobe port and River Muko. The data includes raw sequence reads and associated metadata. This dataset enables comparative analyses of different eDNA concentration methods (glass fiber filters, Sterivex, and QuickConc) and provides insights into the fish community composition in the investigated environments. ### Files and variables #### File: sea.zip **Description:** Contains the MiFish data from the sea water samples.  #### File: river.zip **Description:** Contains the MiFish data from the river water samples. **RawData:** Contains the raw FASTQ files obtained from MiSeq sequencing for each sample. Files are named according to the concentration methods, sample types (1–4, NC), sampleID, sampling sites, and pairs of reads (R1, R2). Name rules are shown below. * {date}-{concentration methods}-{sample types}_{sampleID}_L001_{pairs of reads}_001.fastq.gz **Abbreviations:** * ASV: Amplicon Sequence Variant * BAC: Benzylalkyldimethylammonium chloride (main component of QC1 buffer in QuickConc method) * eDNA: environmental DNA * MiFish: A set of universal PCR primers for fish eDNA metabarcoding * qPCR: quantitative PCR * St: Sterivex * G: Glass fiber filters * SGF: QuickConc * NC: Negative controls ### Code/software Raw data can be analyzed following the materials and methods in the associated paper.