Brute force prey metabarcoding to explore the diets of small invertebrates

1. Prey metabarcoding has become a popular tool in molecular ecology for resolving trophic interactions at high resolution, from various sample types and animals. To date, most predator-prey studies of small-sized animals (<1 mm) have met the problem of overabundant predator DNA in dietary samples by adding blocking primers/peptide nucleic acids. These primers aim to limit the PCR amplification and detection of the predator DNA but may introduce bias to the prey composition identified by interacting with sequences that are similar to those of the predator. 2. Here we demonstrate the use of an alternative method to explore the prey of small marine copepods using whole-body DNA extracts and deep, brute force metabarcoding of an 18S rDNA fragment. 3. After processing and curating raw data from two sequencing runs of varying depth (0.4 and 5.4 billion raw reads), we isolated 1.3 and 52.2 million prey reads, with average depths of ~15 900 and ~120 000 prey reads per copepod individual, respectively. While data from both sequencing runs were sufficient to distinguish dietary compositions from disparate seasons, locations and copepod species, greater sequencing depth led to better separation of clusters. 4. As computation and sequencing are becoming ever more powerful and affordable, we expect the brute force approach to become a general standard for prey metabarcoding, as it offers a simple and affordable solution to consumers that are impractical to dissect or unknown to science.