Analysis of environmental DNA (eDNA) enables identifying the presence of a species of interest in a simple and high-throughput manner. Typically, these analyses are performed by amplifying a short DNA sequence using highly specific primers. Here, we generalized this approach and characterized, by massively parallel sequencing, DNA molecules amplified using primers that are specific of a given taxon, yet provide enough information to identify the species carrying each DNA sequence. We tested this approach by analyzing 90 water samples of 40 mL collected along the Cuyahoga River. We amplified eDNA extracted from each water sample using 13 primer pairs – targeting bacteria, archaea, mammals, fish, algae, amphibians, birds, bryophytes, arthropods, copepods, diatoms, fungi and plants – and simultaneously sequenced all PCR products by high-throughput sequencing. Despite the small sample volumes, we identified DNA sequences from 14 species of fish, 17 species of mammals, 8 species of birds, 15 species of arthropods, one turtle and one salamander. Interestingly, in addition to aquatic and semi-aquatic animal species, we amplified DNA from many terrestrial species that live near the banks of the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that has not been reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small sample of water by universal amplification combined with massively parallel sequencing can provide a broad perspective on the biological diversity of a given environment.
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