16SrRNS gene amplicon by Illumina miseq:  Humpback whale blow
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 V1-V3 region of the bacterial 16S rRNA gene was amplified using barcoded 27F (5'-AGA GTT TGA TCM TGG CTC AG- 3') and 519R primers (5' - GWATTACCGCGGCKGCTG - 3') (Lane 1991; Lane et al. 1985). 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 (2x300 bp) on the Illumina MiSeq platform.