Abstract
Analysis of environmental DNA (eDNA) enables the detection of species of interest from water and soil samples, typically using species-specific PCR. Here, we describe a method to characterize the biodiversity of a given environment by amplifying eDNA using primer pairs targeting a wide range of taxa and high-throughput sequencing for species identification. We tested this approach on 91 water samples of 40 mL collected along the Cuyahoga River (Ohio, USA). We amplified eDNA using 12 primer pairs targeting mammals, fish, amphibians, birds, bryophytes, arthropods, copepods, plants and several microorganism taxa and sequenced all PCR products simultaneously by high-throughput sequencing. Overall, we identified DNA sequences from 15 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 animals, we identified DNA from terrestrial species that live near the Cuyahoga River. We also identified DNA from one Asian carp species invasive to the Great Lakes but that had not been previously reported in the Cuyahoga River. Our study shows that analysis of eDNA extracted from small water samples using wide-range PCR amplification combined with high-throughput sequencing can provide a broad perspective on biological diversity.
Highlights
Environmental samples, such as river and pond water or soil, contain a complex mixture of DNA molecules originating from intact microorganisms, feces, mucous, gametes, shed tissues or decaying parts from organisms living in or near the sampling site[1]
To evaluate the amplification breadth and informativity of “universal” primers we developed PrimerTree, an R package that performs the following functions for each primer pair provided by the user: (1) In silico PCR against a selected NCBI database (2) Retrieval of DNA sequences predicted to be amplified (3) Taxonomic identification of these sequences (4) Multiple DNA sequence alignment (5) Reconstruction of a phylogenetic tree (6) Visualization of the tree with taxonomic annotation
For studies amplifying environmental DNA (eDNA) or DNA from unknown taxa, we require that the amplification works on all members of a given taxon while avoiding off-target amplification
Summary
Environmental samples, such as river and pond water or soil, contain a complex mixture of DNA molecules originating from intact microorganisms, feces, mucous, gametes, shed tissues or decaying parts from organisms living in or near the sampling site[1]. DNA extracted from these samples (often referred to as environmental DNA or eDNA) can be characterized globally by shotgun sequencing all DNA present within a sample This approach (referred to as metagenomics) provides a wealth of information, about the identity of the species present in the environment, and about the gene content of the sequenced organisms which can highlight interesting biological features[2,3]. DNA extracted from environmental samples can be analyzed by PCR targeting a carefully selected locus This approach has been widely applied to ecological studies[4,5,6], conservation[7,8] or to identify the presence of invasive species[9]. Our analyses show that this methodology can provide a broad perspective on the aquatic and terrestrial biodiversity at the sampled sites in a simple, rapid and cost-effective manner
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