Comparison of seven DNA metabarcoding sampling methods to assess diet in a large avian predator

DNA metabarcoding is a rapidly advancing tool for diet assessment in wildlife ecology. Studies have used a variety of field collection methods to evaluate diet, however there is a pressing need to understand the differences among sampling methods and the downstream inferential consequences they may have on our ability to document diet accurately and efficiently. We evaluated seven DNA metabarcoding sampling methods to assess the diet of a large avian predator: Buteo lagopus (rough-legged hawk). We collected beak swabs, talon swabs, cheek (buccal) swabs, cloacal swabs, and cloacal loops from captured birds, and collected fecal samples from both captured and uncaptured birds. We described and compared variation in prey recovery within and among the seven sampling methods and identified appropriate analytical methods to compare diet among individuals sampled via different methods. Beak and talon swabs produced the highest prey detection rates, yielded the greatest prey richness per sample,..., We collected diet samples from Buteo lagopus throughout their North American nonbreeding range in the conterminous United States and Canada during the nonbreeding season (November – March) from 2020 – 2023. We sampled diet from captured birds via: 1) talon swabs, 2) beak swabs, 3) cheek (i.e., buccal cavity) swabs, 4) cloacal swabs, 5) cloacal fecal loops, and 6) fecal samples. We also collected 7) fecal samples from uncaptured free-ranging birds. In total, we collected 592 samples from 189 individuals including 113 captured and 76 uncaptured Buteo lagopus.
All samples were extracted in a laboratory dedicated to low quality and quantity DNA samples.  No forms of high-quality DNA were handled or stored in this laboratory. We collected DNA onto three “substrates” as described above: nylon bristle swabs (beak and talon samples), foam swabs (cheek and cloacal samples) and homogenized feces (fecal samples, fecal loops). Each substrate had its own DNA extraction protocol, but the protoco..., , # Comparison of seven DNA metabarcoding sampling methods to assess diet in a large avian predator

[https://doi.org/10.5061/dryad.rv15dv4hh](https://doi.org/10.5061/dryad.rv15dv4hh)

## Description of the data and file structure

Diet metabarcoding samples were collected using bristle and foam swabs from Rough-Legged Hawk beaks, talons, cheeks, cloacas and feces. DNA from swabs was extracted in a low-input (clean) DNA laboratory in Moscow, ID. Vertebrates were targeted using the V5 region of the 12S RNA gene using primers from Riaz et al (2011). Libraries were prepped using a two-step protocol with the locus PCR in the first round and an i5/i7 dual-index PCR in the second round. Samples were sequenced in duplicate with rolling positive replicates between libraries (ie, one sample from a previous library included in duplicate in a subsequent library to assess inter-library concordance), a duplicate off-target positive control, and a duplicate no template control in each library. Librarie...