Since our understanding of ecosystem functioning depends on the data within a food chain, outdated, biased, or incomplete assessments of diet weaken our ability to predict how and why changes to ecosystems occur. Conventional methods of constructing food chains rely on diet studies which visually identify prey during feeding events or within stomachs, pellets, or feces. These techniques often suffer from MLN4924 Metabolic Enzyme/Protease inhibitor misidentification of similar-looking prey, underrepresentation of soft-bodied or small prey, and low taxonomic resolution, where prey cannot be precisely identified due to distance or digestion. Biochemical methods such as fatty acids, stable isotopes, and DNA have gained popularity in diet studies and can be used as a form of quality control for conventional techniques. However, these methods offer insight on diet in the longer term and are not necessarily well suited for prey species identification, particularly when there is little a priori knowledge of diet. The use of DNA within feces or stomach contents provides a snapshot of Tasocitinib predator diet and has not only been shown to provide a better estimate of diet than conventional methods, but can also provide species-level prey identification through the use of publicly available reference sequences. DNA-based techniques are advantageous in that they can be used either for comparative purposes for previously known diets or for de novo diet description. The application of DNA barcoding in diet studies has increased considerably with the advent of next-generation sequencing technology. It is now possible to identify even the rarest prey from multiple predators to species, genus, or family level in a single sequencing run while maintaining the ability to trace back each prey to the sample from which it came. NGS has been used in barcoding diet studies for fur seals, little penguins, slow worms, bats, leopard cats and tapirs. However prey identification from DNA in diet samples can be greatly influenced by technical issues including the uncertainty about the taxonomic diversity expected in the sample and the poor quality of the genomic DNA, particularly when extracted from fecal samples. Most DNA-based diet studies design multiple group-specific primers that amplify 
the various prey types in predator diet, but these studies may fail to describe the full taxonomic range of the prey consumed. Universal primers can amplify and resolve species across a broad variety of taxa, making them a good, cost- and time-effective alternative to group-specific primers. Using multiple markers may also provide a broader taxonomic resolution of diet as different markers are not suitable barcodes for all taxonomic groups. In addition, PCR amplification of degraded DNA is more reliable when target fragments are small. Moreover, up to 90% of the sequences obtained from NGS can be less-degraded host DNA. The inclusion of primers to block host DNA amplification can increase the number of prey sequences significantly. Finally, it is also possible that the results may be misleading if primers can amplify the prey within the prey on which predators feed. This may skew the interpretation of how those species interact with the rest of the ecosystem. Thus a comparison of the diet of both predator and prey is warranted to assess the potential for detection of secondary consumption by DNA based methods. To date, no diet studies employing NGS have investigated multiple components of a food chain. Here we use Atlantic puffins of Machias Seal Island and their main prey, Atlantic herring as a model system to investigate how molecular methods can be used to describe food chains as these two species represent a simple food web where two conventional methods of studying diet have been employed. Puffin chick diet is known from hundreds of hours of observing adults provisioning their chicks as part of a long-term seabird research program.