Illumina MiSeq library preparation

Sample library preparation used a two-step PCR procedure consisting of ‘amplicon’ and ‘index’ PCR reactions, as described previously [47]. Amplicon PCR was performed by amplifying the ITS1 region of the rRNA gene using BITS and B58S3 primers [48] with CS1 and CS2 linker sequences, respectively. Index PCR primers contained Illumina MiSeq adapter sequences, unique eight nucleotide barcodes, 9–12 bp heterogeneity spacers, and CS1/CS2 linker sequences. After both PCR reactions, samples were submitted to the IBEST Genomics Resources Core at the University of Idaho (Moscow, ID, USA) for quantification, normalization, pooling, and sequencing. Paired-end sequencing (300 bp) was performed on an Illumina MiSeq Desktop Sequencer (Illumina Inc., San Diego, CA, USA).