An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

DNA analysis of predator faeces using high‐throughput amplicon sequencing (HTS) enhances our understanding of predator–prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous an...

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Published in:	Molecular ecology resources Vol. 19; no. 1; pp. 176 - 190
Main Authors:	Jusino, Michelle A., Banik, Mark T., Palmer, Jonathan M., Wray, Amy K., Xiao, Lei, Pelton, Emma, Barber, Jesse R., Kawahara, Akito Y., Gratton, Claudio, Peery, M. Zachariah, Lindner, Daniel L.
Format:	Journal Article
Language:	English
Published:	England Wiley Subscription Services, Inc 01.01.2019
Subjects:	AMPtk animal manures Aquatic insects arthropod mock community Arthropoda Arthropods bat guano Bats Bioinformatics Chiroptera Communities Community Culicidae cytochrome-c oxidase Cytochromes Deoxyribonucleic acid Diet dietary analysis DNA DNA sequencing Dung Economic conditions feces Guano insectivore insectivores Insects Mitochondria mitochondrial genes Mosquitoes next‐generation sequencing oligodeoxyribonucleotides Parameterization Pitfall traps Polymerase chain reaction Predator-prey interactions predator-prey relationships Prey Primers sequence analysis Workflow arthropod mock community AMPtk dietary analysis next-generation sequencing bat guano insectivore
ISSN:	1755-098X, 1755-0998, 1755-0998
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Abstract	DNA analysis of predator faeces using high‐throughput amplicon sequencing (HTS) enhances our understanding of predator–prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair “ZBJ” to results using the novel primer pair “ANML.” To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single‐copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre‐ and post‐PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects 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%). Furthermore, in an HTS comparison of field‐collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.
AbstractList	DNA analysis of predator faeces using high-throughput amplicon sequencing (HTS) enhances our understanding of predator-prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair "ZBJ" to results using the novel primer pair "ANML." To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single-copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre- and post-PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects 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%). Furthermore, in an HTS comparison of field-collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization. DNA analysis of predator faeces using high-throughput amplicon sequencing (HTS) enhances our understanding of predator-prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair "ZBJ" to results using the novel primer pair "ANML." To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single-copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre- and post-PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects 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%). Furthermore, in an HTS comparison of field-collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.DNA analysis of predator faeces using high-throughput amplicon sequencing (HTS) enhances our understanding of predator-prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair "ZBJ" to results using the novel primer pair "ANML." To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single-copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre- and post-PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects 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%). Furthermore, in an HTS comparison of field-collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.
Author	Jusino, Michelle A. Barber, Jesse R. Peery, M. Zachariah Kawahara, Akito Y. Banik, Mark T. Gratton, Claudio Lindner, Daniel L. Pelton, Emma Wray, Amy K. Xiao, Lei Palmer, Jonathan M.
Author_xml	– sequence: 1 givenname: Michelle A. orcidid: 0000-0002-3284-4254 surname: Jusino fullname: Jusino, Michelle A. email: michellejusino@gmail.com, mjusino@ufl.edu organization: University of Florida – sequence: 2 givenname: Mark T. surname: Banik fullname: Banik, Mark T. organization: Center for Forest Mycology Research – sequence: 3 givenname: Jonathan M. orcidid: 0000-0003-0929-3658 surname: Palmer fullname: Palmer, Jonathan M. organization: Center for Forest Mycology Research – sequence: 4 givenname: Amy K. surname: Wray fullname: Wray, Amy K. organization: University of Wisconsin‐Madison – sequence: 5 givenname: Lei surname: Xiao fullname: Xiao, Lei organization: Florida Museum of Natural History, University of Florida – sequence: 6 givenname: Emma surname: Pelton fullname: Pelton, Emma organization: The Xerces Society for Invertebrate Conservation – sequence: 7 givenname: Jesse R. orcidid: 0000-0003-3084-2973 surname: Barber fullname: Barber, Jesse R. organization: Boise State University – sequence: 8 givenname: Akito Y. orcidid: 0000-0002-3724-4610 surname: Kawahara fullname: Kawahara, Akito Y. organization: Florida Museum of Natural History, University of Florida – sequence: 9 givenname: Claudio orcidid: 0000-0001-6262-9670 surname: Gratton fullname: Gratton, Claudio organization: University of Wisconsin‐Madison – sequence: 10 givenname: M. Zachariah surname: Peery fullname: Peery, M. Zachariah organization: University of Wisconsin‐Madison – sequence: 11 givenname: Daniel L. orcidid: 0000-0002-2951-4481 surname: Lindner fullname: Lindner, Daniel L. email: dlindner@fs.fed.us, dlindner@wisc.edu organization: Center for Forest Mycology Research
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Snippet	DNA analysis of predator faeces using high‐throughput amplicon sequencing (HTS) enhances our understanding of predator–prey interactions. However, conclusions... DNA analysis of predator faeces using high-throughput amplicon sequencing (HTS) enhances our understanding of predator-prey interactions. However, conclusions...
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SubjectTerms	AMPtk animal manures Aquatic insects arthropod mock community Arthropoda Arthropods bat guano Bats Bioinformatics Chiroptera Communities Community Culicidae cytochrome-c oxidase Cytochromes Deoxyribonucleic acid Diet dietary analysis DNA DNA sequencing Dung Economic conditions feces Guano insectivore insectivores Insects Mitochondria mitochondrial genes Mosquitoes next‐generation sequencing oligodeoxyribonucleotides Parameterization Pitfall traps Polymerase chain reaction Predator-prey interactions predator-prey relationships Prey Primers sequence analysis Workflow
Title	An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals
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