16S amplicon metabarcoding of marine metagenome: Portsmouth Harbor, NH (USA)
Samples were collected and filtered onto 0.2 um PC filters. DNA was extracted from filters using the ZymoBIOMICS DNA/RNA Miniprep kit. RNA was also extracted from samples. A two-step PCR approach was used, targeting 16S rRNA with primers 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 926R (5-CCGYCAATTYMTTTRAGTTT-3). Primers were constructed with Fluidigm common oligos CS1 forward (CS1-TS-F: 5-ACACTGACGACATGGTTCTACA-3) and CS2 reverse (CS2-TS-R: 5-TACGGTAGCAGAGACTTGGTCT-3) fused to their 5' ends. PCR products were sent to the Michigan State University Research Technology Support Facility Genomics Core for secondary PCR and sequencing. Secondary PCR used dual-indexed, Illumina-compatible primers, targeting the Fluidigm CS1/CS2 oligomers at the ends of the PCR products. Sequencing runs were performed on an Illumina MiSeq to produce 250x250 nt paired reads.