title: DNA sequence of PCR negative control
design: As negative control of PCR, nuclease-free water was added into PCR reaction mix in place of the DNA sample. The forward primer 5.8S_Fun 5- AACTTTYRRCAAYGGATCWCT-3 and the reverse primer ITS4_Fun 5- AGCCTCCGCTTATTGATATGCTTAART-3 were used. The first PCR reaction mix contained nuclease-free water, 1 M of each of the appropriate primers, 0.02 U/l KAPA HiFi HotStart ReadyMix and 2 l of template in a total reaction volume of 15 l. Each sample was made in 8 replicates. The following thermocycling program was used: 5 min of denaturation at 95 C, followed by 30 cycles of 20 s at 98 C, 15 s at 50 C and 15 s at 72 C. Final extension was run at 72 C for 5 min. Next, the replicates were merged and concentrated using the commercial kit Genomic DNA Clean & Concentrator (ZYMO Research, Irvine, CA, USA). The second PCR reaction mix contained nuclease-free water, 1 M of each of the appropriate primers, 0.02 U/l KAPA HiFi HotStart ReadyMix and 1 l of template in a total reaction volume of 25 l. The following thermocycling program was used: 5 min of denaturation at 95 C, followed by 11 cycles of 20 s at 98 C, 15 s at 50 C and 15 s at 72 C. Final extension was run at 72 C for 5 min. Next, amplicons were sent to the Core Facility for Nucleic Acid at the University of Alaska Fairbanks for Illumina Miseq platform sequencing.