16S amplicon metabarcoding of marine metagenome: Bear Cut, FL (USA)
Samples were collected and filtered onto Sterivex 0.22 um cartridge filters. DNA was extracted from Sterivex by adding lysis buffer and beads using different bead sizes and magnetic bead-based extraction kits (Zymo and NucleoMag). Following bead beating in the cartridges, extractions were finished on an automated KingFisher Flex instrument (Thermo Fisher) in 96-well plates. A two-step PCR approach was used, targeting 16S rRNA with primers 515F (5-GTGYCAGCMGCCGCGGTAA-3) and 926R (5-CCGYCAATTYMTTTRAGTTT-3) or 12S rRNA using primers MiFish-U-F (5-GCCGGTAAAACTCGTGCCAGC-3) and MiFish-U-R (5-CATAGTGGGGTATCTAATCCCAGTTTG-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 250+250 nt paired reads.