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...# 七种DNA元条形码（DNA metabarcoding）采样方法评估大型猛禽食性的比较研究 DNA元条形码（DNA metabarcoding）技术是当前野生动物生态学领域用于食性评估的快速演进手段。过往研究已采用多种野外采集方法开展食性评估，但目前亟需明确不同采样方法间的差异，及其对我们精准高效记录食性的能力所产生的后续推断影响。本研究针对大型猛禽——毛脚鵟（Buteo lagopus）的食性，评估了七种DNA元条形码采样方法。我们从捕获个体采集喙部拭子、爪部拭子、颊部（口腔）拭子、泄殖腔拭子及泄殖腔拭环，并从捕获与未捕获个体均采集粪便样本。我们描述并比较了七种采样方法内部及彼此间的猎物检出变异，并确立了适用于比较不同采样方法所获个体食性的分析方法。喙部与爪部拭子的猎物检出率最高，单样本的猎物丰富度也最高……2020至2023年的非繁殖季（11月至次年3月），我们在毛脚鵟的北美非繁殖分布区（美国本土及加拿大境内）采集其食性样本。我们通过以下六种方式采集捕获个体的食性样本：1）爪部拭子、2）喙部拭子、3）颊部（即口腔）拭子、4）泄殖腔拭子、5）泄殖腔粪便拭环、6）粪便样本；此外还从7）未捕获的野生个体采集粪便样本。总计，我们从189只毛脚鵟个体采集了592份样本，其中113只为捕获个体，76只为未捕获个体。 所有样本均在专门处理低质量、低浓度DNA样本的实验室中完成提取，该实验室未处理或储存任何形式的高质量DNA。如前文所述，我们将DNA收集于三类"载体"：尼龙鬃拭子（对应喙部与爪部样本）、泡沫拭子（对应颊部与泄殖腔样本）及匀浆粪便（对应粪便样本、粪便拭环样本）。每类载体均配有专属的DNA提取流程，但流程…… [https://doi.org/10.5061/dryad.rv15dv4hh](https://doi.org/10.5061/dryad.rv15dv4hh) ## 数据与文件结构说明 本研究通过鬃拭子与泡沫拭子，从毛脚鵟的喙部、爪部、颊部、泄殖腔及粪便中采集食性元条形码样本。拭子样本的DNA提取工作在爱达荷州莫斯科市的低投入（洁净）DNA实验室完成。本研究采用Riaz等人2011年开发的引物，靶向12S RNA基因（12S RNA gene）的V5区域以检测脊椎动物。文库构建采用两步法流程：第一轮进行位点PCR扩增，第二轮进行i5/i7双索引PCR（i5/i7 dual-index PCR）扩增。所有样本均进行双份测序，并在文库间设置滚动阳性重复对照（即从前一批文库中选取一份样本，在后续文库中以双份形式纳入，以评估文库间的一致性）；每个文库均设置双份脱靶阳性对照及双份无模板对照。文库……