title: Sensitivity and accuracy of high-throughput metabarcoding methods for early detection of invasive fish species.
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.
authors.affiliation: Mid-Continent Ecology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, Environmental Protection Agency, Duluth, MN, 55804, United States of America.|Oak Ridge Institute for Science and Education Participant, Oak Ridge, TN, 37831, United States of America.
authors.forename: Chelsea
authors.initials: C
authors.lastname: Hatzenbuhler
authors.affiliation: Mid-Continent Ecology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, Environmental Protection Agency, Duluth, MN, 55804, United States of America.
authors.forename: John R
authors.initials: JR
authors.lastname: Kelly
authors.affiliation: Exposure Methods and Measurements Division, National Exposure Research Laboratory, Office of Research and Development, Environmental Protection Agency, Cincinnati, OH, 45268, United States of America.
authors.forename: John
authors.initials: J
authors.lastname: Martinson
authors.affiliation: Oak Ridge Institute for Science and Education Participant, Oak Ridge, TN, 37831, United States of America.|Systems Exposure Division, National Exposure Research Laboratory, Office of Research and Development, Environmental Protection Agency, Cincinnati, OH, 45268, United States of America.
authors.forename: Sara
authors.initials: S
authors.lastname: Okum
authors.affiliation: Systems Exposure Division, National Exposure Research Laboratory, Office of Research and Development, Environmental Protection Agency, Cincinnati, OH, 45268, United States of America.
authors.forename: Erik
authors.initials: E
authors.lastname: Pilgrim
mesh_terms: D000818:Animals; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D003576:Electron Transport Complex IV / Q000235:genetics*; D029941:Fish Proteins / Q000235:genetics; D005399:Fishes / Q000145:classification* / Q000196:embryology / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D058865:Introduced Species; D057230:Limit of Detection; D056186:Metagenomics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D029941:Fish Proteins; D003576:Electron Transport Complex IV