DOI: 10.5061/dryad.54v2q
title: Data from: MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species

description: We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding
 environmental DNA (eDNA) from fishes. Primers were designed using aligned
 whole mitochondrial genome (mitogenome) sequences from 880 species,
 supplemented by partial mitogenome sequences from 160 elasmobranchs
 (sharks and rays). The primers target a hypervariable region of the 12S
 rRNA gene (163–185 bp), which contains sufficient information to identify
 fishes to taxonomic family, genus and species except for some closely
 related congeners. To test versatility of the primers across a diverse
 range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi
 Aquarium with known species compositions, prepared dual-indexed libraries
 and performed paired-end sequencing of the region using high-throughput
 next-generation sequencing technologies. Out of the 180 marine fish
 species contained in the four tanks with reference sequences in a custom
 database, we detected 168 species (93.3%) distributed across 59 families
 and 123 genera. These fishes are not only taxonomically diverse, ranging
 from sharks and rays to higher teleosts, but are also greatly varied in
 their ecology, including both pelagic and benthic species living in
 shallow coastal to deep waters. We also sampled natural seawaters around
 coral reefs near the aquarium and detected 93 fish species using this
 approach. Of the 93 species, 64 were not detected in the four aquarium
 tanks, rendering the total number of species detected to 232 (from 70
 families and 152 genera). The metabarcoding approach presented here is
 non-invasive, more efficient, more cost-effective and more sensitive than
 the traditional survey methods. It has the potential to serve as an
 alternative (or complementary) tool for biodiversity monitoring that
 revolutionizes natural resource management and ecological studies of fish
 communities on larger spatial and temporal scales.
description: Script for extracting
 DNA sequences flanked by MiFish primer-match regions.For each of the primer
 pair, the region with the smallest hamming distance is found in each DNA
 sequence. If the smallest hamming distance is larger than 3, such
 sequences are ignored because existence of indels or many mutations is
 assumed in primer-match regions. For those ignored sequences, manual
 extraction needs to be conducted afterwards.hamming_match.rbAnalytical pipeline for DNA sequences amplified by MiFish primersThe MiFish pipeline works on the Linux/Unix environment. In the first step, the pipeline conducts quality-filtering of the MiFish sequence reads, including Phred score-based low-quality tail-trimming, length filtering, deletion of N base-containing reads, paired-end read assembly, and primer sequences removal. The fasta files of the reads that passed through these quality check are generated. As the next step, the pipeline performs sequence similarity-based species-assignment and the generation of a species by read-count table. This tab-spaced table provides species diversity information detected from the MiFish PCR meta-barcoding analysis of environmental DNAs.MiFish_pipeline.zip