title: 16Sv4 community amplicon seq of Atta texana fungus garden: d3e10
design: 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), 0.4 mM of each dNTP (Promega), 0.5 _M of each Illumina-indexed primer, a 0.05 _M spike-in of non-indexed primers, 0.4 _g/_L BSA (NEB), and 2.5 mM MgCl2, to which NFW was added to a volume of 50 _L. Primers 515F-Parada and 806R-Apprill. This volume was split into triplicate reactions for thermocycling using conditions: 30 s at 98 C, 30 cycles of 10 s at 98 C, 30 s at 55 C, and 30 s at 72 C, followed by a 2 min final extension at 72C. Triplicates were then pooled. Amplified libraries of a similar concentration were pooled together at equal concentrations. Mag-Bind TotalPure NGS beads (OMEGA) were used to remove small DNA fragments from the pooled libraries using a 1:0.8 volume ratio of library to beads. An additional two-sided size bead cleanup removed remaining  fragments from non-specific amplication: Fragments  600 bp  were removed using 1:0.65 ratio, then fragments  100bp were removed with a 1:0.8 ratio. 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.