DOI: 10.5061/dryad.6gd51
title: Data from: A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents

description: Introduction: The PCR-based analysis of homologous genes has become one of
 the most powerful approaches for species detection and identification,
 particularly with the recent availability of Next Generation Sequencing
 platforms (NGS) making it possible to identify species composition from a
 broad range of environmental samples. Identifying species from these
 samples relies on the ability to match sequences with reference barcodes
 for taxonomic identification. Unfortunately, most studies of environmental
 samples have targeted ribosomal markers, despite the fact that the
 mitochondrial Cytochrome c Oxidase subunit I gene (COI) is by far the most
 widely available sequence region in public reference libraries. This is
 largely because the available versatile (“universal”) COI primers target
 the 658 barcoding region, whose size is considered too large for many NGS
 applications. Moreover, traditional barcoding primers are known to be
 poorly conserved across some taxonomic groups. Results: We first design a
 new PCR primer within the highly variable mitochondrial COI region, the
 “mlCOIintF” primer. We then show that this newly designed forward primer
 combined with the “jgHCO2198” reverse primer to target a 313 bp fragment
 performs well across metazoan diversity, with higher success rates than
 versatile primer sets traditionally used for DNA barcoding (i.e.
 LCO1490/HCO2198). Finally, we demonstrate how the shorter COI fragment
 coupled with an efficient bioinformatics pipeline can be used to
 characterize species diversity from environmental samples by
 pyrosequencing. We examine the gut contents of three species of
 planktivorous and benthivorous coral reef fish (family: Apogonidae and
 Holocentridae). After the removal of dubious COI sequences, we obtained a
 total of 334 prey Operational Taxonomic Units (OTUs) belonging to 14 phyla
 from 16 fish guts. Of these, 52.5% matched a reference barcode
 (&gt;98% sequence similarity) and an additional 32% could be assigned
 to a higher taxonomic level using Bayesian assignment. Conclusions: The
 molecular analysis of gut contents targeting the 313 COI fragment using
 the newly designed mlCOIintF primer in combination with the jgHCO2198
 primer offers enormous promise for metazoan metabarcoding studies. We
 believe that this primer set will be a valuable asset for a range of
 applications from large-scale biodiversity assessments to food web
 studies.
description: Fasta formatted file
 containing all unique COI sequences obtained from fish gut
 contents454 platform was used
 for sequencing the gut content of 16 coral reef fishes. Unique sequences
 were obtained after initial denoising using pyronoise (implemented in
 Mothur) and alignment to the BIOCODE reference dataset. More details about
 sequence processing are available in the method section of the
 manuscriptNocturnal.fastaSummary table of gut content DNA sequencingThe table presents the number of sequences per OTU and per sample (fish gut). Sequence clustering was done using CROP. OTUs were identified from BLASTn searches performed in the Moorea BIOCODE database and GENBANK. We provide BIOCODE specimen number and/or GenBank accession number in the table whenever sequence similarity to a reference barcode was &gt;98%. Additional information on BIOCODE specimen can be obtained from http://biocode.berkeley.edu/. When sequence similarity was &lt;98%, we used the Bayesian assignment tool implemented in SAP to assign each OTU to a higher taxonomic group, accepting assignments at a significance level of 95% (posterior probability). Note that the representative sequence for each OTU is provided as a supplementary file in the manuscript.Nocturnal_OTUs_summary.xlsx