Who is eating what: diet assessment using next generation sequencing

Abstract

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

Flower preferences and pollen transport networks for cavity‐nesting solitary bees: Implications for the design of agri‐environment schemes

“…One of the main advantages of DNA based methodology is the sensitivity of the technique to very small quantities of DNA; however, this also makes very low‐level contamination in natural samples hard to exclude. We have attempted to use very conservative criteria in the identifications; however, the presence–absence nature of metabarcoding analyses (Pompanon et al., 2012) can also overemphasize the importance of rare or trace material. Despite these cautions, this study provides tentative evidence that R. canina might be a previously unknown forage plant for these bee species.…”

Discovering hidden biodiversity: the use of complementary monitoring of fish diet based on DNA barcoding in freshwater ecosystems

“…The studies available using these more advanced methods such as NGS of stomach content of fecal material in food‐web studies provided more powerful biodiversity estimates (Pompanon et al. 2012; De Barba et al. 2014).…”

“…Previous studies based on DNA analyses have principally focused on gut content analysis, or on food web composition (Pompanon et al. 2012; De Barba et al. 2014); however, none of these studies have examined the effectiveness of the procedure for monitoring or evaluating biodiversity.…”

Feeding selectivity of bivalve larvae on natural plankton assemblages in the Western English Channel

“…Next generation sequencing (NGS) allows sequencing of several thousands of sequences simultaneously, increasing the efficiency and decreasing the cost and time associated with more traditional amplification, cloning and sequencing techniques. DNA metabarcoding using NGS has the potential to reveal many consumed species simultaneously (O'Rorke et al 2012;Pompanon et al 2012;Lindeque et al 2013). In addition, the most recent studies have developed a method whereby the dietary analysis of consumers can be made with universal primers using predator-specific endonuclease restriction enzymes or blocking primers, including the analysis of small planktonic consumers such as bivalve larvae (Maloy et al 2013).…”