Abstract
High-throughput DNA metabarcoding has gained recognition as a potentially powerful tool for biomonitoring, including early detection of aquatic invasive species (AIS). DNA based techniques are advancing, but our understanding of the limits to detection for metabarcoding complex samples is inadequate. For detecting AIS at an early stage of invasion when the species is rare, accuracy at low detection limits is key. To evaluate the utility of metabarcoding in future fish community monitoring programs, we conducted several experiments to determine the sensitivity and accuracy of routine metabarcoding methods. Experimental mixes used larval fish tissue from multiple “common” species spiked with varying proportions of tissue from an additional “rare” species. Pyrosequencing of genetic marker, COI (cytochrome c oxidase subunit I) and subsequent sequence data analysis provided experimental evidence of low-level detection of the target “rare” species at biomass percentages as low as 0.02% of total sample biomass. Limits to detection varied interspecifically and were susceptible to amplification bias. Moreover, results showed some data processing methods can skew sequence-based biodiversity measurements from corresponding relative biomass abundances and increase false absences. We suggest caution in interpreting presence/absence and relative abundance in larval fish assemblages until metabarcoding methods are optimized for accuracy and precision.
Highlights
Fish species[14], but monitoring larval fish communities may provide some benefits over adult surveillance because detecting new invaders in larval form would more strongly suggest the presence of a successfully reproducing population posing an imminent threat
Due to the challenges associated with traditional taxonomic identification of larval fishes, monitoring larval fish communities for early detection of invasive fish species may only be practical if an alternative identification method is employed
c oxidase subunit I (COI) sequences for target A, P. semilunaris were recovered in 50% and 25% of replicates with target biomass representing 0.1% (T3) and 0.02% (T5) of total biomass, respectively and positive detection was attributed to 1–5 sequences per hit
Summary
Fish species[14], but monitoring larval fish communities may provide some benefits over adult surveillance because detecting new invaders in larval form would more strongly suggest the presence of a successfully reproducing population posing an imminent threat. Due to the challenges associated with traditional taxonomic identification of larval fishes, monitoring larval fish communities for early detection of invasive fish species may only be practical if an alternative identification method is employed. High-throughput sequencing (HTS), or metabarcoding, enables simultaneous sequencing of a high-resolution genetic marker (DNA barcode) in many samples (multiplexing) providing a fast, and potentially cost-effective method for estimating biodiversity in multi-species assemblages[25,26,27]. PCR design, and downstream sequence data processing methods influence taxonomic resolution and accuracy of the final biodiversity estimates[31,32,33,34,35]. Using larval fish as a relevant life stage, we carried out several experiments designed to investigate the sensitivity and accuracy of metabarcoding methods commonly used to characterize composition of samples with a mixture of species from the larval fish community
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