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\begin{document}

\title{New tools for diet analysis: nanopore sequencing of metagenomic DNA from
rat stomach contents to quantify diet}



\author[1]{Nikki Freed}%
\author[1]{William Pearman}%
\author[1]{Adam Smith}%
\author[1]{Georgia Breckell}%
\author[1]{James Dale}%
\author[1]{Olin Silander}%
\affil[1]{Massey University - Albany Campus}%


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  \date{May 25, 2024}


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\begin{abstract}
Accurate determination of animal diets is difficult. Methods such as
molecular barcoding or metagenomics offer a promising approach, allowing
quantitative and sensitive detection of different taxa. Here we show
that rapid and inexpensive diet quantification is possible through
metagenomic sequencing with the portable Oxford Nanopore Technologies
(ONT) MinION. Using an amplification-free approach, we profiled the
stomach contents from 24 wild-caught rats. We conservatively identified
diet items from over 50 taxonomic orders, ranging across nine phyla,
including plants, vertebrates, invertebrates, and fungi. This highlights
the wide range of taxa that can be identified using this simple
approach. We calibrated the accuracy of this method by comparing the
characteristics of reads matching the ground-truth host genome (rat) to
those matching diet items, and show that at the family-level, taxon
assignments are approximately 97.5\% accurate. Some inaccuracies may
arise from database biases; we suggest a way to mitigate for database
biases when using metagenomic approaches. Finally, we implemented a
constrained ordination analysis and show that we can identify the
sampling location of an individual rat within tens of kilometres based
on diet content alone. This work establishes proof-of-principle for
long-read metagenomic methods in quantitative diet analysis. We show
that diet content can be quantified even with limited expertise, using a
simple, amplification free workflow and a relatively inexpensive and
accessible next generation sequencing method. Continued increases in the
accuracy and throughput of ONT sequencing, along with improved genomic
databases, suggests that a metagenomic approach for quantification of
animal diets will become an important method in the future.%
\end{abstract}%



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