## 2 Methodology
### DNA Extraction and Next-Generation Sequencing  
Firstly, water samples (500 mL each from five sites) were collected from the selected regions in the Bay of Bengal. These samples were then sent to the laboratory and concentrated. The DNA extraction process (the DNA kit was formulated to isolate genomic DNA from water samples) continued about 3-4 h, then bacterial cells were collected. The concentration and purity of the template DNA were measured by a spectrophotometer). On average, 270 ng/\(\mu\)L of DNA were extracted from each sample and placed directly on the spectrophotometer.  
The following steps were used for DNA extraction, following the method by [26]:  
* a tube (2 mL) containing glass beads (\(\omega\)0.1 and \(\omega\)0.5 mm) was selected to store the water samples (membranes in 70% ethanol);
* suspension was centrifuged (at 8000 g) for 10 minutes;
* after centrifugation, the supernatant was redundant and the pellet was re-proscribed in pure water (200 \(\mu\)L);
* the glass bead tube containing the membrane received resuspension;
* phenol-chloroform protocol was followed to perform the DNA extraction process as previously described by [13];
* isopropanol containing 1 \(\mu\)L glycogen precipitated the template DNA at \(-\)20 \({}^{\circ}\)C for 30 minutes;
* the pellet was washed with 70% ethanol after centrifugation at 15,000 g for 15 minutes;
* after drying, the template DNA was finally dissolved in 30 \(\mu\)L TE buffer, and the concentration and purity of the template DNA were measured by a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA) then stored at \(-\)20 \({}^{\circ}\)C for further analysis.  
The bacterial concentration was calculated by considering all the dilution steps during the sample preparation and expressed as copies number per mL. A \(t\)-test was applied to determine the significance difference of the total 16S rRNA gene copy number in the water. The V3 hypervariable region of the 16S rRNA gene was amplified by nested PCR.  
Only primers with an overall coverage above 75% for Bacteria were considered for primer pair analysis. All primers are available in probeBase, a comprehensive online database for rRNA-targeted oligonucleotides (www.microbial-ecology.net/probebase). PCR primer pairs had previously been validated in the literature [27,28], and we used 341F- 5'CCTACGGGGAGGCACAG3' and 534R-5'ATTACCGCGCTGCTGCG3'.  
Figure 1: Bacterial diversity (as number) over the Bay of Bengal region, Bangladesh. The highest and lowest numbers of bacteria are shown as red and blue, respectively.  
Next-generation sequencing protocol were been followed for preparing the gene libraries, and the samples were quantified by the real-time quantitative PCR at a 95% confidence level. The preparation for this experiment followed the study of [12], and the concentration of bacteria was calculated following the dilution steps during the preparation of samples.  
The V3-V4 hypervariable region of the bacterial 16S rRNA gene (460-bp size on average) was amplified using primers. Each water bottle contained a distinct community structure at the phylum, class, genus and operational taxonomic unit (OTU) levels one day after bottling, and were class-dominated by \(\gamma\)-Proteobacteria. At the genus level, the dominant genera were Acinetobacter. A total of 141,497 qualified reads (ranging from 19,241 to 34,323 per DNA sample) were recovered. The predominant phylum was the Proteobacteria (43.6% of total 16s rRNAgene sequences).  
On the Illumina MiSeq platform, samples were paired-end sequenced (low-quality sequences were removed) at a read length of 300 nt. Amplification and sequencing were achieved under negative control.