Addressing nontarget amplification in DNA metabarcoding studies of arthropod-feeding rodents.

High-throughput sequencing approaches have revolutionized how we study animal diets by enabling the detection of dietary components from the metabarcoding of DNA in excrement. Mitochondrial cytochrome oxidase C subunit I (mtCOI) DNA metabarcoding is commonly used to study the diets of arthropod-feeding animals; however, this approach is susceptible to nontarget amplification of the consumer species mtCOI locus. Nontarget amplification is often an unforeseen complication that can drastically reduce the quality and utility of the results generated by high-throughput amplicon sequencing. By interrogating the diets of new world rodents in the genus (woodrats) in both natural and captive settings, we demonstrate that nontarget amplification can drastically reduce the total read abundance of detected arthropod taxa in fecal samples and inhibit downstream analyses of dietary diversity and composition metrics. Using the results from these investigations, we offer a guide on how to identify concerns for nontarget amplification when selecting degenerate primers for DNA metabarcoding studies and recommend several approaches that can reduce or eliminate nontarget amplification. Lastly, for the community interested in investigating the diets of arthropod-feeding rodents, we generated a database containing the degree of mismatch between publicly available Rodentia mtCOI sequences and four common universal mtCOI primer sets to be used as a resource for inferring the relative risk of nontarget amplification when designing arthropod metabarcoding studies in rodent systems. This guide will be especially useful for researchers working with consumer species that have not previously been studied.