Fungal ITS amplicon sequencing of food product
To enable efficient multiplexing, we employed a two-step amplicon protocol that first amplifies the marker gene using primers that include Nextera adapter tails, then using this as template for a second round of PCR to add unique indices and Illumina flow cell adapters (Gohl et al. 2016). First, the internal transcribed spacer 2 ("ITS2") region of the ribosomal RNA cistron was PCR-amplified using the primer pairs 5.8S-Fun_Nextera and ITS4-Fun_Nextera, which consist of the primers 5.8S-Fun and ITS4-Fun (Taylor et al. 2016) with Illumina Nextera adapter tails. Sequencing libraries were prepared from these amplicons by using a 1:99 dilution as template for a second round of PCR with indexing primers consisting of Nextera adapter, unique 8-bp index, and Illumina flow cell adapter. The resulting DNA was then cleaned and normalized across all samples using the AxyPrep Mag^TM PCR Normalizer Protocol (Axygen Biosciences, Union City, CA).