title: Metabarcoding of mitochondrial 16S targeting fishes from eDNA extracted from a deionized water negative control at a stream sampling location in Great Smoky Mountains National Park.
design: 1L of deionized water was taken into the field and filtered at the streamside. eDNA from this field negative control was used for library Library prep for MiSeq sequencing followed the strategy of the Illumina 16S protocol (Illumina Part # 15044223 Rev. B), where a universal tail was added to the primers to allow incorporation of Nextera indices in an indexing PCR step. Field negative controls were only sequenced with the fish 16S marker. Prior to index PCR, each sample was amplified in triplicate and pooled to reduce stochastic amplification variation among replicates. Up to 96 samples were sequenced in a single run.