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Jusino MA, Banik MT, Palmer JM, Wray AK, Xiao L, Pelton E, Barber JR, Kawahara AY, Gratton C, Peery MZ, Lindner DL.2017. An improved method for utilizing high-throughput amplicon sequencing to determine the diets of insectivorous animals. PeerJ Preprints5:e3184v1https://doi.org/10.7287/peerj.preprints.3184v1
DNA analysis of predator feces 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 (CO1). 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) CO1 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 of 59 taxa (41%). Furthermore, in an HTS comparison of field-collected samples, the ANML primers detected nearly four-fold 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.
This manuscript is currently under review at Molecular Ecology Resources and the submission of this preprint was approved by the editorial board of Molecular Ecology Resources.
Appendix S1 – DNA extraction details for the CLS extraction for arthropods