title: DNA metabarcoding diet analysis
design: The 12S rRNA region was amplified using the primers Teleo-12S-F 5'-ACTGGGATTAGATACCCCACT-3' and Teleo-12S-R 5'-TCACAGGGTAAGCTGACGAC-3', to which the Illumina sequencing primer sequences were attached to their 5 ends. PCRs were carried out in a final volume of 25 L, containing approximately 50 ng of template DNA, 0.5 M of each forward and reverse primer, 12.5 L of Phusion High-Fidelity PCR Master Mix (Thermo Scientific), and ultrapure water up to 25 L. The reaction mixture was incubated as follows: an initial denaturation at 98 C for 30 s, followed by 35 cycles of 98 C for 10 s, 60 C for 20 s, 72 C for 20 s, and a final extension step at 72 C for 10 minutes. Samples were individually tagged in the second PCR round with identical conditions but only 5 cycles and 60 C as the annealing temperature. Libraries were pooled in equimolar amounts into a single pool for sequencing in a fraction of an Illumina's MiSeq PE300 run.