ITS-2 metabarcoding of Cyathostominae: mock communityM1
ITS-2 gene region was PCR amplified using the NC1 and NC2 primers, added of one to three random bases to the 5' primer end, a 2bp linker and 28 bp Illumina adapter overhang. Single multiplexing was performed using a homemade 6 bp index, which was added to reverse primer during a second PCR with 12 cycles using forward primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC) and reverse primer (CAAGCAGAAGACGGCATACGAGAT-index-GTGACTGGAGTTCAGACGTGT).