DOI: 10.5061/dryad.kwh70rz4s
title: The precautionary principle and dietary DNA metabarcoding: commonly used abundance thresholds change ecological interpretation

description: Dietary DNA metabarcoding enables researchers to identify and characterize
 trophic interactions with a high degree of taxonomic precision. It is also
 sensitive to sources of bias and contamination in the field and lab. One
 of the earliest and most common strategies for dealing with such
 sensitivities has been to filter resulting sequence data to remove
 low-abundance sequences before conducting ecological analyses based on the
 presence or absence of food taxa. Although this step is now often
 perceived to be both necessary and sufficient for cleaning up datasets,
 evidence to support this perception is lacking and more attention needs to
 be paid to the related risk of introducing other undesirable errors. Using
 computer simulations, we demonstrate that common strategies to remove
 low-abundance sequences can erroneously eliminate true dietary sequences
 in ways that impact downstream dietary inferences. Using real data from
 well-studied wildlife populations in Yellowstone National Park, we further
 show how these strategies can markedly alter the composition of individual
 dietary profiles in ways that scale-up to obscure ecological
 interpretations about dietary generalism, specialism, and niche
 partitioning. Although the practice of removing low-abundance sequences
 may continue to be a useful strategy to address a subset of research
 questions that focus on a subset of relatively abundant food resources,
 its continued widespread use risks generating misleading perceptions about
 the structure of trophic networks. Researchers working with dietary DNA
 metabarcoding data—or similar data such as environmental DNA, microbiomes,
 or pathobiomes—should be aware of potential drawbacks and consider
 alternative bioinformatic, experimental, and statistical solutions.
description: We used fecal DNA metabarcoding to characterize the diets of
 bison and bighorn sheep in winter and summer. Our analyses are based on 35
 samples (median per species per season = 10) analyzed using the P6 loop of
 the chloroplast <i>trn</i>L(UAA) intron together with publicly
 available plant reference data (Illumina sequence read data are available
 at NCBI (BioProject: PRJNA780500)). <i>Obicut</i> was used to
 trim reads with a minimum quality threshold of 30, and primers were
 removed from forward and reverse reads using <i>cutadapt</i>.
 All further sequence identifications were performed using obitools;
 forward and reverse sequences were aligned using the
 <i>illuminapairedend </i>command using a minimum alignment
 score of 40, and only joined sequences retained. We used the
 <i>obiuniq</i> command to group identical sequences and tally
 them within samples, enabling us to quantify the relative read abundance
 (RRA) of each sequence. Sequences that occurred ≤2 times overall or that
 were ≤8 bp were discarded. Sequences were considered to be likely PCR
 artifacts if they were highly similar to another sequence (1 bp
 difference) and had a much lower abundance (0.05%) in the majority of
 samples in which they occurred; we discarded these sequences using the
 <i>obiclean</i> command. Overall, we
 characterized 357 plant sequences and a subset of 355 sequences were
 retained in the dataset after rarefying samples to equal sequencing depth.
 We then applied relative read abundance thresholds from 0% to 5% to the
 fecal samples. We compared differences in the inferred dietary richness
 within and between species based on individual samples, based on average
 richness across samples, and based on the total richness of each
 population after accounting for differences in sample size.
description: The readme file contains an explanation of each of the variables
 in the dataset. Information on the methodology can be found in the
 associated manuscript referenced above.