Dataset Information

Efficacy of metabarcoding for identification of fish eggs evaluated with mock communities.

ABSTRACT: There is urgent need for effective and efficient monitoring of marine fish populations. Monitoring eggs and larval fish may be more informative than that traditional fish surveys since ichthyoplankton surveys reveal the reproductive activities of fish populations, which directly impact their population trajectories. Ichthyoplankton surveys have turned to molecular methods (DNA barcoding & metabarcoding) for identification of eggs and larval fish due to challenges of morphological identification. In this study, we examine the effectiveness of using metabarcoding methods on mock communities of known fish egg DNA. We constructed six mock communities with known ratios of species. In addition, we analyzed two samples from a large field collection of fish eggs and compared metabarcoding results with traditional DNA barcoding results. We examine the ability of our metabarcoding methods to detect species and relative proportion of species identified in each mock community. We found that our metabarcoding methods were able to detect species at very low input proportions; however, levels of successful detection depended on the markers used in amplification, suggesting that the use of multiple markers is desirable. Variability in our quantitative results may result from amplification bias as well as interspecific variation in mitochondrial DNA copy number. Our results demonstrate that there remain significant challenges to using metabarcoding for estimating proportional species composition; however, the results provide important insights into understanding how to interpret metabarcoding data. This study will aid in the continuing development of efficient molecular methods of biological monitoring for fisheries management.

SUBMITTER: Duke EM

PROVIDER: S-EPMC7141059 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Efficacy of metabarcoding for identification of fish eggs evaluated with mock communities.

Duke Elena M EM Burton Ronald S RS

Ecology and evolution 20200303 7

There is urgent need for effective and efficient monitoring of marine fish populations. Monitoring eggs and larval fish may be more informative than that traditional fish surveys since ichthyoplankton surveys reveal the reproductive activities of fish populations, which directly impact their population trajectories. Ichthyoplankton surveys have turned to molecular methods (DNA barcoding & metabarcoding) for identification of eggs and larval fish due to challenges of morphological identification. ...[more]

PMID: 32274002

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Project description:1: Metabarcoding (high-throughput sequencing of marker gene amplicons) has emerged as a promising and cost-effective method for characterizing insect community samples. Yet, the methodology varies greatly among studies and its performance has not been systematically evaluated to date. In particular, it is unclear how accurately metabarcoding can resolve species communities in terms of presence-absence, abundances, and biomass. 2: Here we use mock community experiments and a simple probabilistic model to evaluate the effect of different DNA extraction protocols on metabarcoding performance. Specifically, we ask four questions: (Q1) How consistent are the recovered community profiles across replicate mock communities?; (Q2) How does the choice of lysis buffer affect the recovery of the original community?; (Q3) How are community estimates affected by differing lysis times and homogenization?; and (Q4) Is it possible to obtain adequate species abundance estimates through the use of biological spike-ins? 3: We show that estimates are quite variable across community replicates. In general, a mild lysis protocol is better at reconstructing species lists and approximate counts, while homogenization is better at retrieving biomass composition. Small insects are more likely to be detected in lysates, while some tough species require homogenization to be detected. Results are less consistent across biological replicates for lysates than for homogenates. Some species are associated with strong PCR amplification bias, which complicates the reconstruction of species counts. Yet, with adequate spike-in data, species abundance can be determined with roughly 40% standard error for homogenates, and with roughly 50% standard error for lysates, under ideal conditions. In the latter case, however, this often requires species-specific reference data, while spike-in data generalizes better across species for homogenates. 4: We conclude that a non-destructive, mild lysis approach shows the highest promise for presence/absence description of the community, while also allowing future morphological or molecular work on the material. However, homogenization protocols perform better for characterizing community composition, in particular in terms of biomass.

Project description:Metabarcoding of environmental DNA is increasingly used for biodiversity assessments in aquatic communities. The efficiency and outcome of these efforts are dependent upon either de novo primer design or selecting an appropriate primer set from the dozens that have already been published. Unfortunately, there is a lack of studies that have directly compared the efficacy of different metabarcoding primers in marine and estuarine systems. Here we evaluate five commonly used primer sets designed to amplify rRNA barcoding genes in fishes and compare their performance using water samples collected from estuarine sites in the highly biodiverse Indian River Lagoon in Florida. Three of the five primer sets amplify a portion of the mitochondrial 12S gene (MiFish_12S, 171bp; Riaz_12S, 106 bp; Valentini_12S, 63 bp), one amplifies 219 bp of the mitochondrial 16S gene (Berry_16S), and the other amplifies 271 bp of the nuclear 18S gene (MacDonald_18S). The vast majority of the metabarcoding reads (> 99%) generated using the 18S primer set assigned to non-target (non-fish) taxa and therefore this primer set was omitted from most analyses. Using a conservative 99% similarity threshold for species level assignments, we detected a comparable number of species (55 and 49, respectively) and similarly high Shannon’s diversity values for the Riaz_12S and Berry_16S primer sets. Meanwhile, just 34 and 32 species were detected using the MiFish_12S and Valentini_12S primer sets, respectively. We were able to amplify both bony and cartilaginous fishes using the four primer sets with the vast majority of reads (>99%) assigned to the former. We detected the greatest number of elasmobranchs (six species) with the Riaz_12S primer set suggesting that it may be a suitable candidate set for the detection of sharks and rays. Of the total 76 fish species that were identified across all datasets, the combined three 12S primer sets detected 85.5% (65 species) while the combination of the Riaz_12S and Berry_16S primers detected 93.4% (71 species). These results highlight the importance of employing multiple primer sets as well as using primers that target different genomic regions. Moreover, our results suggest that the widely adopted MiFish_12S primers may not be the best choice, rather we found that the Riaz_12S primer set was the most effective for eDNA-based fish surveys in our system.

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Efficacy of metabarcoding for identification of fish eggs evaluated with mock communities.

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Efficacy of metabarcoding for identification of fish eggs evaluated with mock communities.

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