title: 16SrRNS  gene amplicon by Illumina miseq:  Humpback whale blow
design: Nucleic acids were extracted  from cotton swabs and in the case of seawater from filtering units using the  FastDNA Spin Kit for Soil following the manufacturers protocol (MP  Biomedicals, Santa Ana, California, USA). Blank DNA extractions were  performed to account for contamination in DNA extraction reagents. The V4  region of the bacterial 16S rRNA gene was amplified using barcoded 515F  (5-GTG BCA GCM GCC GCG GTA A-3) and 806R primers (5-GGA CTA CHV GGG TAT  CTA ATC C-3) (Caporaso et al., 2012). Each sample was amplified in  duplicates. The final PCR reaction volume was 25 l consisting of 0.125 l of  each primer (40M, Integrated DNA Technologies, Coralville, IA, USA), 5 l of  5X Green GoTaq Flexi Buffer (Promega, Madison, WI, USA), 0.3125 l of Ex Taq  HotStart Version (5 Units per l, TaKaRa, Bio Inc., Shiga, Japan), 2 l of  dNTP-mix (10 mM, TaKaRa, Bio Inc., Shiga, Japan), 5 l of MgCl2 (25 mM;  Promega, Madison, WI, USA) and 4 l of DNA. Negative (no DNA template) and  positive controls (Escherichia coli genomic DNA) were included. Reactions  were performed using a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad  Laboratories Inc, Hercules, CA, USA) under the following program conditions:  Initial denaturation at 98C for 2 min; then 35 cycles of denaturation at  98C for 10 s, annealing at 50C for 30 s and elongation at 72C for 60 s;  and a final elongation at 72C for 5 min. PCR products were submitted to the  Ramaciotti Centre for Genomics (Sydney, Australia) for purification, library  preparation and paired-end amplicon sequencing (2x250 bp) on the Illumina  MiSeq platform.