Metabarcoding of mitochondrial 16S targeting aquatic invertebrates from eDNA extracted from a freshwater stream sediment sample within Great Smoky Mountains National Park.
Equal aliquots of extracted eDNA from three replicate stream bottom sediment scoop grabs collected at the stream site were combined, and amplified using a 16S primer pair targeting aquatic invertebrates. 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. 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.