Received: 30 March 2017 | Revised: 1 August 2018 | Accepted: 19 September 2018 DOI: 10.1111/1755-0998.12951 RESOURCE ARTICLE An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals Michelle A. Jusino1,2* | Mark T. Banik1* | Jonathan M. Palmer1 | Amy K. Wray3 | Lei Xiao4 | Emma Pelton5,6 | Jesse R. Barber7 | Akito Y. Kawahara4 | Claudio Gratton5 | M. Zachariah Peery3 | Daniel L. Lindner1 1United States Department of Agriculture, Forest Service, Northern Research Station, Abstract Center for Forest Mycology Research, DNA analysis of predator faeces using high‐throughput amplicon sequencing (HTS) Madison, Wisconsin enhances our understanding of predator–prey interactions. However, conclusions 2Department of Plant Pathology, University of Florida, Gainesville, Florida drawn from this technique are constrained by biases that occur in multiple steps of 3Department of Forest and Wildlife the HTS workflow. To better characterize insectivorous animal diets, we used DNA Ecology, University of Wisconsin‐Madison, Madison, Wisconsin from a diverse set of arthropods to assess PCR biases of commonly used and novel 4McGuire Center for Lepidoptera and primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We Biodiversity, Florida Museum of Natural compared diversity recovered from HTS of bat guano samples using a commonly History, University of Florida, Gainesville, Florida used primer pair “ZBJ” to results using the novel primer pair “ANML.” To parame- 5Department of Entomology, University of terize our bioinformatics pipeline, we created an arthropod mock community con- Wisconsin‐Madison, Madison, Wisconsin sisting of single‐copy (cloned) COI sequences. To examine biases associated with 6The Xerces Society for Invertebrate Conservation, Portland, Oregon both PCR and HTS, mock community members were combined in equimolar 7Department of Biological Sciences, amounts both pre‐ and post‐PCR. We validated our system using guano from bats Graduate Program in Ecology, Evolution fed known diets and using composite samples of morphologically identified insects and Behavior, Boise State University, Boise, Idaho collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%), whereas ZBJ amplified 24–40 of 59 taxa (41%–68%). Further- Correspondence MichelleA.Jusino,DepartmentofPlant more, in an HTS comparison of field‐collected samples, the ANML primers detected Pathology,UniversityofFlorida,Gainesville,FL. nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods Emails:[email protected]; [email protected] detected include medically and economically relevant insect groups such as mosqui- and toes. Results revealed biases at both the PCR and sequencing levels, demonstrating DanielL.Lindner,UnitedStatesDepartmentof Agriculture,ForestService,NorthernResearch the pitfalls associated with using HTS read numbers as proxies for abundance. The Station,CenterforForestMycologyResearch, use of an arthropod mock community allowed for improved bioinformatics pipeline OneGiffordPinchotDrive,Madison,WI. Emails:[email protected];[email protected] parameterization. Funding information KEYWORDS United States Forest Service, Northern ‐ Research Station; University of Wisconsin‐ AMPtk, arthropod mock community, bat guano, dietary analysis, insectivore, next generation Madison, Wisconsin Agricultural Experiment sequencing Station, Grant/Award Number: Hatch Formula Funds; National Science Foundation, Grant/Award Number: IOS- 1121739, IOS-1121807 *Indicates shared first authorship based on equal contributions. 176 | © 2018 John Wiley & Sons Ltd wileyonlinelibrary.com/journal/men Mol Ecol Resour. 2019;19:176–190. JUSINO ET AL. | 177 1 | INTRODUCTION Rydell et al., 2016; Vesterinen et al., 2016) and birds (Crisol‐Martínez et al., 2016; Jedlicka et al., 2016; Trevelline et al., 2016). Although High‐throughput amplicon sequencing (HTS) has become the pre- the ZBJ primers have been widely utilized, there are indications that ferred method for rapid molecular identification of members of they have a narrow taxonomic range (Brandon‐Mong et al., 2015; mixed ecological communities. HTS is now also increasingly used to Clarke et al., 2014; Mallott et al., 2015). identify the arthropod dietary components of a wide taxonomic Along with primer choice, many other assumptions and parame- range of animals including mammals (Bussche et al., 2016; Clare ters commonly employed in HTS environmental DNA analyses have et al., 2014; Clare, Symondson, & Fenton, 2014; Mallott, Malhi, & a large impact on the operational taxonomic units (OTUs) that are Garber, 2015; Rydell et al., 2016; Vesterinen et al., 2016), birds (Cri- recovered and appropriate positive controls are necessary to deter- sol‐Martínez, Moreno‐Moyano, Wormington, Brown, & Stanley, mine those impacts. Bioinformatics clustering algorithms can influ- 2016; Jedlicka, Vo, & Almeida, 2016; Trevelline, Latta, Marshall, Nut- ence apparent diversity within a sample, or an entire library of tle, & Porter, 2016), reptiles (Kartzinel & Pringle, 2015), fish (Harms‐ samples, and trimming and filtering parameters can impact the result- Tuohy, Schizas, & Appeldoorn, 2016) and arthropods (Krehenwinkel, ing community composition (Deagle, Thomas, Shaffer, Trites, & Jar- Kennedy, Pekár, & Gillespie, 2016). Identification of the DNA of man, 2013). A validation or control is needed to accurately dietary components is accomplished by “metabarcoding,” which parameterize bioinformatics pipelines; therefore, the use of mock involves extracting DNA from faecal samples, amplifying one or communities as positive controls in HTS is increasingly becoming more barcoding loci, preparing DNA libraries and finally sequencing, common, especially among researchers who work with fungal and bioinformatics and data analysis. Each of these steps involves deci- bacterial communities (Bokulich & Mills, 2013; Bokulich et al., 2013; sions and assumptions that significantly affect results. For example, Nguyen et al., 2015; Palmer, Jusino, Banik, & Lindner, 2018). Mock biases are unavoidable when amplifying environmental DNA with communities can be used to examine biases, starting at the sampling PCR‐based methods (Brooks et al., 2015) and careful consideration step and ending at the bioinformatics and community analysis steps. should be exercised when selecting a primer pair for HTS. Thus, Here, we provide a comprehensive assessment of an improved while DNA metabarcoding is a powerful tool for studying trophic molecular and analytical pipeline for molecular‐based arthropod diet interactions, conclusions should take into account the shortcomings determination. We used a reference arthropod community to iden- and parameters of the techniques (e.g., Brooks et al., 2015; D'Amore tify specific amplification biases associated with three commonly et al., 2016; Lindahl et al., 2013; Nguyen, Smith, Peay, & Kennedy, used primer pairs, including ZBJ, and two novel primer pairs, LCOI‐ 2015; Pompanon et al., 2012). 1490/COI‐CFMRa (hereafter ANML) and LCOI‐1490/COI‐CFMRb The mitochondrial cytochrome oxidase C subunit 1 locus (COI) is (hereafter CFMRb), for the COI region (Table 1). To further test pri- the most frequently used barcoding locus for identifying a wide mers, we compared HTS results from the ZBJ primers to our novel range of taxonomic groups, including arthropods. Because COI has ANML primer pair using field‐collected bat guano samples. We the most extensive reference library for arthropods (BOLD systems, designed an arthropod mock community based on single‐copy Ratnasingham and Hebert, 2007), it is the most commonly used (cloned) mitochondrial COI sequences, which can serve as a standard locus for dietary studies of insectivorous animals (Clarke, Soubrier, in HTS sequencing and to help parameterize a bioinformatics pipe- Weyrich, & Cooper, 2014). The entire COI barcoding region is about line. Finally, we validated the accuracy of our system of novel pri- 658 base pairs (bp) and currently too long to be used efficiently with mers, the mock community control and our bioinformatics pipeline most HTS platforms. Therefore, it is necessary to sequence shorter using guano from bats fed known insect diets and composite sam- regions of the COI locus, which has proven challenging due to a lack ples of morphologically identified arthropods from pitfall traps. of conserved priming sites within the COI region (Deagle, Jarman, Coissac, Pompanon, & Taberlet, 2014). Therefore, novel primer pairs 2 | METHODS AND MATERIALS should be tested against as many expected target DNA sequences as possible. 2.1 | Testing of primer pairs against known insect Zeale, Butlin, Barker, Lees, and Jones (2011) developed the ZBJ‐ samples ArtF1c/ZBJ‐ArtR2c (hereafter ZBJ) primer pair for detecting arthro- pod prey DNA in bat guano by amplifying a 157‐bp fragment of the DNA was extracted from 59 arthropod taxa belonging to 12 orders COI region. In the initial study (Zeale et al., 2011), which employed following the protocol in Lindner and Banik (2009) with modifica- cloning and sequencing rather than HTS, the ZBJ primers amplified tions for insects (Supporting Information Appendix S1). Briefly, DNA 37 taxa from 13 arthropod orders but did not amplify bat COI DNA. was extracted from excised leg muscles of larger insects, or for smal- The ZBJ primers were designed to target a short fragment to amplify ler insects, the thorax was punctured and the entire insect