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

Abstract

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.

A total crapshoot? Evaluating bioinformatic decisions in animal diet metabarcoding analyses

“…A smaller gene fragment (~180 bp) of each specimen was amplified using the ANML primers described in Jusino et al. (2019). These PCR products were pooled in equimolar ratios for use in our experiments.…”

“…Mock communities can be used to assess systematic error and biases in observed sequence data (Gohl et al., 2016), optimize filtering parameters (Bokulich et al., 2013), understand tradeoffs among sequence error correction approaches (Nearing, Douglas, Comeau, & Langille, 2018), and evaluate taxonomic classification regimes (Bokulich, Kaehler, et al, 2018). Systematic evaluation of animal metabarcoding studies is growing but remain limited in scope; synthetic mock samples have been used to explore the potential for alternative primer use (Beng et al., 2016), while biological mock samples have been used to improve quality filtering of spurious sequence variants (Jusino et al., 2019) and to evaluate the utility of PCR replicates (Galan et al., 2018). In addition, few studies have used real data (i.e., actual diet samples) to offer insights into the effects of sequencing platforms (Divoll, Brown, Kinne, McCracken, & O'Keefe, 2018) or abundance filtering parameters (Alberdi et al., 2018).…”

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

“…We increased the base degeneracy level of the COI reverse primer described in Jusino et al. (2018) and of the 16S primers from Epp et al. (2012), to improve primer binding during PCR amplification.…”

“…Metabarcoding analysis of the DNA contained in bat guano has thus been developed (a) to deepen our understanding of bat ecology (Arrizabalaga‐Escudero et al., 2015; Clare, Symondson, Broders, et al, 2014), (b) to highlight the potential ecosystem services provided by bats as pest suppressors (Aizpurua et al., 2018; Maslo et al., 2017; Wray et al., 2018), and ultimately, (c) to set up effective conservation strategies (Arrizabalaga‐Escudero et al., 2015). However, most of these studies have used specific arthropod primer sets (but see Galan et al., 2018; Jusino et al., 2018; Wray et al., 2018) or specific bat primer sets (Walker, Williamson, Sanchez, Sobek, & Chambers, 2016). While working with guano collected from roost sites, a potential problem arises from the fact that several bat species may roost in the same sites.…”

Untitled

“…Additionally, the primers used (ZBJ−ArtF1c + ZBJ−ArtR2c) by Alberdi et al (2017) performed poorly in our study. This particular primer combination (Zeale et al 2011) is widely used for metabarcoding studies (Jusino et al 2018) The use of COI primer sets with limited or no degeneracy such as in (Zhang et al 2018;Jusino et al 2018) is not recommended. In general, careful primer design and validation (ideally using mock communities) cannot be replaced by the use of multiple COI primer sets (Alberdi et al 2017;Zhang et al 2018) or ribosomal markers (Deagle et al 2014), given the increased workload of a multi marker/primer approach and the limited taxonomic resolution of ribosomal markers (Clarke et al 2017;Marquina et al 2018).…”