Evaluating metabarcoding to analyse diet composition of species foraging in anthropogenic landscapes using Ion Torrent and Illumina sequencing

Abstract

DNA metabarcoding of faecal samples is being successfully used to study the foraging niche of species. We assessed the ability of two benchtop high-throughput sequencing (HTS) platforms, to identify a large taxonomic array of food items from domestic cats Felis silvestris catus, including prey and human-related food taxa (pet food and leftovers leaving undetectable solid remains in faeces). Scats from a captive feeding trial (n = 41) and from free-ranging individuals (n = 326) were collected and analysed using a cytb mini-barcode in independent PCR replicates on the Ion PGM and the MiSeq platforms. Outputs from MiSeq were more sensitive and reproducible than those from Ion PGM due to a higher sequencing depth and sequence quality on MiSeq. DNA from intact prey taxa was detected more often (82% of the expected occurrences) than DNA from pet food (54%) and raw fish and meat (31%). We assumed that this variability was linked to different degree of DNA degradation: The Ion PGM detected significantly less human-linked food, birds, field voles, murids and shrews in the field-collected samples than the MiSeq platform. Pooling the replicates from both platforms and filtering the data allowed identification of at least one food item in 87.4% of the field-collected samples. Our DNA metabarcoding approach identified 29 prey taxa, of which 25 to species level (90% of items) including 9 rodents, 3 insectivores, 12 birds and 1 reptile and 33 human-related food taxa of which 23 were identified to genus level (75% of items). Our results demonstrate that using HTS platforms such as MiSeq, which provide reads of sufficiently high quantity and quality, with sufficient numbers of technical replicates, is a robust and non-invasive approach for further dietary studies on animals foraging on a wide range of food items in anthropogenic landscapes.

Food from faeces: Evaluating the efficacy of scat DNA metabarcoding in dietary analyses

“…In order to determine the reliability of sDNA metabarcoding, several controlled experimental studies have been conducted to examine the potential strengths and weaknesses. These studies have mainly scrutinized the specificity and sensitivity of PCR assays [17,18], library preparation and sequencing technologies [19][20][21], impact of environmental factors on scats [22], biological and physiological status of the defecator [22,23]. Few sDNA studies have empirically tested the effectiveness of sDNA metabarcoding in large felids, (but see [10]), and therefore drawing general conclusions from different taxa may introduce bias in result interpretation.…”

A rapid insect species identification system using mini‐barcode pyrosequencing

“…Such mini‐barcodes have already been applied for biodiversity discovery, forensic identification, and old museum specimens . Further, this technique can be used to authenticate endangered species for conservation and to monitor illegal food for safety and traceability …”

“…In this study, we combine pyrosequencing with the GenBank database to make an extendable species identification system. Next‐generation sequencing with mini‐barcodes has been developed for bulk specimen identification and diet composition of species analysis . By combining high‐throughput sequencing techniques, our 16S rDNA mini‐barcode primer set will be helpful for biodiversity discovery and forensic identification.…”

DNA metabarcoding in diet studies: Unveiling ecological aspects in aquatic and terrestrial ecosystems

“…However, generally, these studies are mostly descriptive, and only about one third (47 out 150) addressed impacts of human‐mediated changes in diets (37% of the studies on terrestrial organisms and 22% of the studies on aquatic taxa, see Table S1). For terrestrial species, most of these studies focused on the impacts of urbanized areas (Bohmann et al, ; Coghlan et al, ; Cravens et al, ; Forin‐Wiart et al, ; Groom et al, ; Khanam, Howitt, Mushtaq, & Russell, ; Lim, Ramli, Bhassu, & Wilson, ; Quéméré et al, ; Smith, Thomas, Levi, Wang, & Wilmers, ; Sullins et al, ) and landscape changes due to agriculture (e.g., Branco et al, ; Clare et al, , ; Sugimoto et al, ) in trophic interactions. Importantly, the results obtained so far provide substantial knowledge toward effective conservation planning and management, by identifying the impacts of introduced species, both revealing competition with native fauna (Brown et al, ; Egeter, Bishop, & Robertson, ; Robeson II et al, ; Zarzoso‐Lacoste et al, ) and uncovering the importance of introduced plant species to the diet of endangered species (Ando et al, ).…”

“…Traditional DNA barcoding approaches are dependent on specifically designed primers that target the genomes under investigation (Fišer Pečnikar & Buzan, 2014). In this regard, the use of the cytochrome c oxidase subunit 1 (COI) as a suitable molecular marker for the barcode for animal life is well established (Coissac et al, 2016;Hebert et al, 2003;Lobo et al, 2013), and this was the most widely used marker in the dietary studies surveyed herein (Figure 2a; 67 out of 213 of markers and types of markers, i.e., genomic approaches and microsatellites; Table S1).…”