The effect of PCR replication on species richness estimates using environmental DNA and 16S rRNA vertebrate metabarcoding

eDNA is a relatively new but promising tool for characterizing biological communities. Currently, the two most common eDNA analyses used by biologists are qPCR and metabarcoding. qPCR is more established, using primers to target a single species. Metabarcoding targets multiple species using one primer set. This allows metabarcoding to more broadly characterize community assemblages and potentially detect rare or low-density species. As a new technology, several methodological questions need to be answered. For example, standard protocol uses only a fraction of DNA extracted from a sample, in PCR reactions. Are rare species thus going undetected? To address this, we examined whether we might find different species in one versus two PCR replicates from the same eDNA sample. Duplicate 4L water samples were collected from seven sites on Fossil Creek, AZ. Samples were filtered, DNA was extracted, PCR was performed in duplicate using primers that amplified a hypervariable region of the 16S rRNA gene that can be used to identify vertebrate species. Sequencing was performed on a Illumina MiSeq FGX Forensic Genomics System. Sequence analysis identified 14 vertebrate species, including six fish, six mammals, and two herpetofauna. The most common species were found in every PCR replicate, whereas sequences associated with rare species were often found in only one of the two PCR replicates. POSTER PRESENTATION URI SIG GRANT AND ARIZONA SPACE GRANT AWAR