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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Vydáno v:	Molecular ecology resources Ročník 19; číslo 1; s. 176 - 190
Hlavní autoři:	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.
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	England Wiley Subscription Services, Inc 01.01.2019
Témata:	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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Shrnutí:	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.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23
ISSN:	1755-098X 1755-0998 1755-0998
DOI:	10.1111/1755-0998.12951