Secondary predation constrains DNA-based diet reconstruction in two threatened shark species

The DNA extracts from each gut sample were amplified, tagged separately, and then pooled for sequencing. Two group-specific mini-barcode primers were selected for the amplification of teleost and crustacean DNA, targeting 12S (MiFish: Miya et al., 2015) and 16S (Crust16S short: Berry et al., 2017) mitochondrial DNA genes, respectively. We also used a universal 18S primer set (Zhan et al., 2013) targeting the hypervariable V4 region of the nuclear small subunit ribosomal DNA to amplify templates from a broader fraction of marine metazoans. Polymerase chain reaction (PCR) was performed using the AmpliTaq Gold 360 protocol and thermocycling conditions recommended in (Taberlet, Bonin, Zinger, & Coissac, 2018). The PCR hybridization temperatures were 50, 51 and 50 oC for MiFish, Crust16S, and Uni18S primer sets, respectively, and products were run on a 1% agarose gel to confirm amplification of the correct target size (MiFish = ±170 bp; Crust16S = ±170 bp, Uni18S = ±220 bp). A second round of PCR was undertaken on the cleaned PCR products using unique dual-indexed primers for each sample, which included the Illumina-specific sequencing adaptors. PCR products were sent to the Ramaciotti Centre for Genomics at the University of New South Wales for cleaning, normalising, and pooling prior to paired-end sequencing, which was performed using a 500 cycle MiSeq V3 Reagent Kit on an Illumina MiSeq platform (Illumina, San Diego, CA, USA). Sample demultiplexing based on the incorporated indexes was conducted by the sequencing centre. Berry, T. E., Osterrieder, S. K., Murray, D. C., Coghlan, M. L., Richardson, A. J., Grealy, A. K., et al. (2017). DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea). Ecology and Evolution, 7(14), 5435–5453. Miya, M. et al. MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species. Roy. Soc. Open Sci. 2(7), 150088 (2015). Taberlet, P., Bonin A., Zinger L., Coissac E. Environmental DNA for Biodiversity Research and Monitoring. Oxford, UK. Oxford University Press (2018). Zhan, A. et al. High sensitivity of 454 pyrosequencing for detection of rare species in aquatic communities. Methods in Ecology and Evolution, 4(6), 558–565 (2013).