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

By Matthieu Leray, Joy Y. Yang, Christopher P. Meyer, S. Mills, N. Agudelo, Vincent Ranwez, J. T. Boehm, Ryuji J. Machida. Frontiers in Zoology, 2013

1,421 citations recorded by the source corpus.

IntroductionThe 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.ResultsWe 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 (>98% sequence similarity) and an additional 32% could be assigned to a higher taxonomic level using Bayesian assignment.ConclusionsThe 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.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/735ebff73f278d1b6c4e4185ea9f77b0cd13e4e0

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Citations in the corpus

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment (2017, positive): “For example, the mlCOIint primers which have a maximum degeneracy of two nucleotides at each position (Leray et al., 2013), were previously tested with two mock communities and up to 35% of taxa remained undetected (Leray and Knowlton, 2015).”
Genetic and Manual Survey Methods Yield Different and Complementary Views of an Ecosystem (2017, neutral): “…loci [16S, ca. 116 bp, (Kelly et al., 2016; O’Donnell et al., 2016; Shelton et al., 2016), targeting metazoans exclusively; COI, ca. 315 bp, (Leray et al., 2013), targeting eukaryotes], and Frontiers in Marine Science | www.frontiersin.org 2 January 2017 | Volume 3 | Article 283 one…”
Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples (2018, background): “even with a degenerate version of the Folmer forward primer (jgLCO1490), amplification success of the 5ʹ portion of the barcoding region has been very inefficient with at most 64% of amplification success (Leray et al. 2013).”
MACSE v2: Toolkit for the Alignment of Coding Sequences Accounting for Frameshifts and Stop Codons (2018, neutral): “yses (Leray et al. 2013), and phylogenomic pipelines (Bragg”
A cost‐efficient and simple protocol to enrich prey DNA from extractions of predatory arthropods for large‐scale gut content analysis by Illumina sequencing (2017, background): “When the predator and prey are sufficiently genetically divergent, blocking primers can be designed to prevent the PCR amplification of prey DNA (Vestheim& Jarman 2008; Leray et al. 2013).”
Non‐specific amplification compromises environmental DNA metabarcoding with COI (2019, background): “Addressing this problem with bias requires incorporating a high degree of degeneracy into COI primers (Leray et al., 2013; Marquina, Andersson, & Ronquist, 2019), particularly by the use of multiple inosine sites (Elbrecht & Leese, 2017; Shokralla et al.”
Scrutinizing key steps for reliable metabarcoding of environmental samples (2018, methodology): “, 2012), Leray (Geller, Meyer, Parker, & Hawk, 2013; Leray et al., 2013) and Zeale (Zeale, Butlin, & Barker, 2011).”
Use of DNA metabarcoding for stomach content analysis in the invasive lionfish Pterois volitans in Puerto Rico (2016, methodology): “The specific region of COI is adequately represented in online databases for Caribbean coral reef fishes and invertebrates (Leray et al. 2013), as well as estimates of relative abundance of species in benthic samples (Leray & Knowlton 2015).”
Phylogeography unplugged: comparative surveys in the genomic era (2014, background): “Emerging metagenomic approaches are now taking advantage of such barcode libraries to evaluate community diversity (e.g., Leray et al 2013).”