DNA metabarcoding diet analysis
The rbcL chloroplast region was amplified using the primers Diat_rbcL 708F_2 (5' AGG TGA AGT TAA AGG TTC WTA YTT AAA 3') and R3_1 (5' CCT TCT AAT TTA CCW ACW ACT G 3'), to which the Illumina sequencing primer sequences were attached to their 5' ends. PCRs were prepared in a volume of 25 uL, containing 1 uL of template DNA, 0.5 uM of the primers, 6.25 uL of Supreme NZYTaq 2x Green Master Mix (NZYTech), and ultrapure water up to 25 uL. The thermal cycling profile included an initial denaturation at 95 C for 5 min, followed by 35 cycles of 95 C for 30 s, 53 C for 30 s, 72 C for 30 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 an Illumina's MiSeq PE300 run.