COI metabarcoding of Cyathostomin: H2O, Negative control
COI primers consisted of a 24-bp long forward (5-RGCHAARCCNGGDYTRTTRYTDGG-3) and 25-bp long reverse (5-GYTCYAAHGAAATHGAHCTHCTHCG-3) primers, and a random single, double or triple nucleotide was added to the 5 primer end to promote sequence complexity and avoid signal saturation. A 28-bp Illumina overhang was added for the forward and reverse sequences respectively, for subsequent ligation with Illumina adapters. PCR parameters were 95C for 3 min, followed by a pre-amplification with 5 cycles of 98C for 15 s, 45C for 30 s, 72C for 30 s, followed by 35 cycles of 98C for 15 s, 55C for 30 s, 72C for 30 s then a final extension of 2 min at 72C. A homemade six-bp index was added to the reverse primer during a second PCR with 12 cycles using forward primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC) and reverse primer (CAAGCAGAAGACGGCATACGAGAT-index-GTGACTGGAGTTCAGACGTGT) for single multiplexing. LIbraries were processed in a single run of MiSeq sequencing with 500 cycles.