design: The V4 region of the 16srRNA gene was amplified from each sample using the dual-indexing sequencing strategy (2). Sequencing or amplicons was performed using the Illumina MiSeq platform, with MiSeq Reagent Kit V2 (500 cycles) according to the manufacturer’s instructions except where noted.  Accuprime High Fidelity Taq was used instead of Accuprime Pfx supermix.  A low biomass amplification protocol was used for library construction consisting of 95°C for 2 min followed by 20 cycles of 95°C for 20 sec., 60°C for 15 sec (dropping 0.3°C every cycle), and 72°C for 5 min.  This was followed by 20 cycles of 95°C for 20 sec., 55°C for and 72°C for 5 min., and finishing with 72°C for 10 min.
library_construction_protocol: MiSeq