New tools for diet analyses: nanopore sequencing of metagenomic DNA from stomach contents to quantify diet in an invasive population of rats

Introduced species of mammals in New Zealand have had catastrophic effects on populations of diverse native species. Quantifying the diets of these omnivorous and predatory species is critical for understanding which native species are most impacted, and to prioritize which mammal species and locations should be targeted with control programmes. A variety of methods have been applied to quantify diet components in animals, including visual inspection of gut contents (Daniel 1973; Pierce and Boyle 1991), stable isotope analysis (Major et al. 2007; Carreon-Martinez and Heath 2010), and time-lapse video (Brown and Brown 1997; Dunlap and Pawlik 1996). Increasingly, DNA-based metabarcoding methods are being used (King et al. 2008; Soininen et al. 2009). These metabarcoding methods require a PCR step using primers that bind to highly conserved genomic regions (e.g. mitochondrial COI) to amplify specific regions for sequencing. This step introduces significant bias, primarily due to the lack of a universal primer set (King et al. 2008). Here we show that direct metagenomic sequencing using the Oxford Nanopore Minion allows rapid quantification of rat diets. Using a sample of rats collected from within 100km of Auckland, NZ, we show that these rats consume a wide variety of plant, invertebrate, vertebrate, and fungal taxa, with substantial differences in diet content between locales. We then show that, based on diet content alone, it is possible to pinpoint the sampling location of an individual rat within tens of kilometres. We expect that the rapidly increasing accuracy and throughput of nanopore-based sequencing, as well as increases in the species diversity of genomic databases, will soon allow rapid and unbiased assessments of animal diets in field settings.