Two microliters of PCR grade water were used in PCR with the MiFish 12S primers targeting fishes (F- GCCGGTAAAACTCGTGCCAGC; R- CATAGTGGGGTATCTAATCCCAGTTTG). 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 unique dual 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.
Two microliters of sample were used in PCR with the MiFish 12S primers targeting fishes. 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.