ITS2 community amplicon seq of Trachymyrmex septentrionalis fungus garden: d5e21
10 _L of template DNA was added to 10 _L 5_ Q5 HotStart High-Fidelity Master Mix (NEB), 0.02 units/_L of Q5 Hot Start High-Fidelity DNA Polymerase (NEB), 0.5 _M of each Illumina-indexed primer, a 0.05 _M spike-in of non-indexed primers, 0.16 _g/_L BSA (NEB), and 0.4 mM MgCl2, 50 _M of each dNTP, to which NFW was added to a volume of 50 _L. Primers fITS7 and ITS4 were used. This volume was split into triplicate reactions for thermocycling using conditions: 3 min at 98 C, 30 cycles of 30 s at 98 C, 45 s at 55 C,  and 45 s at 72 C, followed by a final extension for 5 min at 72 C. Triplicates were then pooled. Amplified libraries of a similar concentration were pooled together at equal concentrations. AMPure XP beads beads were used to remove small DNA fragments from the pooled libraries using a 1:0.8 volume ratio of library to beads. Cleaned library pools were concentrated by drying at 45 C for ~1h using a SpeedVac and then resuspended in 15 _L of NFW. Equimolar amounts of each concentrated library pool were combined into a final library adjusted to 4-5 nM and sequenced with an Illumina Miseq V2, 2 _ 250 bp reads.