MiFish 12S amplicon metabarcoding of seawater metagenome: Gulf of Mexico (USA)
Samples were collected and filtered onto 47 mm diameter mixed cellulose ester (MCE) filters with a 0.45 m pore size. DNA was extracted from filters using a phenol-chloroform protocol modified from Renshaw et al. (2015). A two-step PCR approach was used, targeting 12S V5 -V6 rRNA with the MiFish primers MiFish_U fwd: 5-GTCGGTAAAACTCGTGCCAGC-3 and MiFish_U rev; 5-CATAGTGGGGTATCTAATCCCAGTTTGT-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.