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

Abstract

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.

Effect of marker choice and thermal cycling protocol on zooplankton DNA metabarcoding studies

“…We compared OTUs detected with the COI marker from amplicons generated with either (1) the published touchdown PCR protocol (Leray et al., 2013) or (2) a single annealing temperature (46°C) and a reduced number of cycles (amplicons sequenced on the same MiSeq run). Despite similar numbers of reads per sample (mean ± SD = 14,700 ± 4,000 and 15,600 ± 2,800 for 46°C and touchdown protocols, respectively), 254 OTUs representing 105 taxa were detected from the January samples using the 46°C annealing temperature, compared to only 200 OTUs (96 taxa) using the touchdown protocol (Table 2).…”

“…The first round was either (A) the touchdown protocol as per Leray et al. (2013), namely 94°C for 10 min, a 16 cycle touchdown phase (62–1°C per cycle), followed by 25 cycles with an annealing temperature of 46°C (total of 41 cycles), and a final extension at 72°C for 5 min, or (B) the same protocol using 35 cycles with a single annealing temperature (46°C). Three replicate first round PCRs were performed for each DNA extract with each thermal cycling protocol.…”

“…Although not applied directly to zooplankton, Leray and Knowlton (2015) and Leray et al. (2013) used a new COI primer set to characterize marine benthic communities and fish diet, highlighting its potential for assessing marine metazoan biodiversity.…”

“…To compare performance of the three markers, we evaluated taxonomic coverage and resolution, correspondence between morphology‐ and DNA‐based identification, and the ability to assess relative abundance of calanoid copepods from the proportion of HTS reads. For the COI marker, high annealing temperatures in the first rounds of the published touchdown PCR protocol (Leray et al., 2013) could bias PCR amplification toward taxa with less mismatches in the primer‐binding sites (Sipos et al., 2007); hence, we compared the number of taxa detected using the touchdown protocol to a protocol with a single low annealing temperature. We also explored the technical repeatability of taxon detection by re‐sequencing COI amplicons generated with identical PCR protocols.…”

Discovering hidden biodiversity: the use of complementary monitoring of fish diet based on DNA barcoding in freshwater ecosystems

“…One of the few investigations examining the potential of using DNA analyses of fish gut contents in the monitoring of ecosystem function is the study of metabarcoding metazoan diversity of coral reef fish (Leray et al. 2013). Previous studies based on DNA analyses have principally focused on gut content analysis, or on food web composition (Pompanon et al.…”