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.