title: ITS2 metabarcoding of fine roots of Diphasiastrum complanatum
design: The amplification of the ITS2 region as a barcode was conducted using primers gITS7F (Ihnmark et al. 2012) and ITS4ngsR (Tedersoo et al. 2014), each incorporating Illumina overhang adapters (underlined): 5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGARTCATCGARTCTTTG-3 and 5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTCCTSCGCTTATTGATATGC-3, respectively. The PCR was carried out in a 25 l reaction mix comprising 12.5 l of Taq 2x Master Mix RED with 1.5mM MgCl2 (Amplicon, Odense, Denmark), 0.2 M of each primer, and 70 ng of DNA. The PCR protocol included the following steps: (i) 5 min at 94C; (ii) 25 cycles of 30 s at 94C, 30 s at 48C, and 30 s at 72C; (iii) 7 min at 72C. All amplicon libraries were sequenced in a single run using an Illumina MiSeq v3 reagent kit for 2  300 bp paired-end reads (Illumina, San Diego, CA, USA).