Illumina sequences of the ITS2 region of the herbarium specimen
Purified DNA samples were analyzed using a high sensitivity gel on a 5300 Fragment Analyzer to visualize the degree of DNA fragmentation.  The ITS2 region was amplified using Fluidigm reagents and barcodes in a two-step PCR procedure.  In the first step, the ITS2 region was amplified for 35 cycles using the fITS7 plus CS1 Fluidigm primer pad and the ITS4 plus CS2 Fluidigm primer pad.  The second step consisted of 14 cycles attaching the 10 bp sample specific identification barcode and Illumina adaptor barcodes.  The final amplicon construct consisted of the following: Illumina i5 adaptor - CS1 - fITS7 primer - region of interest - ITS4 primer - CS2 - sample specific 10 bp barcode - Illumina i7 adaptor.  The 5300 Fragment Analyzer was used to visualize the amplicons after each PCR step to confirm the presence of amplicons.  The final amplicons were sequenced on an Illumina MiSeq v3 platform rapid 2x250 nt PE NANO v2 paired-end reads.