title: 16S profile freshwater sponge and water
design: Polymerase chain reaction (PCR) was performed following published protocols from the Earth Microbiome Project (http://www.earthmicrobiome.org/protocols-and-standards/).  PCR reactions were run using barcoded 16S rRNA primers (515F and 806R: (Caporaso et al., 2011; Walters et al., 2015).  Barcoded PCR products were cleaned using AMPure beads (Beckman Coulter), and concentrations of the cleaned products were measured using a Qubit fluorometer.  Cleaned products were diluted to 4 nM and then pooled in equal volumes.  Sample preparation and loading onto the Illumina MiSeq followed a standard Illumina protocol, with the exception of using custom sequencing primers from the EMP protocol.  Samples were sequenced using a 500 cycle V2 chemistry kit (Illumina) for 16S gene sequencing to obtain 250bp single-end amplicons.