## 2 Materials and Methods
### DNA Extraction and Metagenomic Sequencing  
Genomic DNA was extracted by means of PowerSoil(r) DNA Isolation Kit (Cat. No. 12888, MO BIO, Germantown, MD, USA) in compliance with the manufacturers' protocol. Extracted DNA for sequencing was prepared according to the Illumina 18S Metagenomic Sequencing Library protocols (San Diego, CA, USA). DNA quantity, quality, and integrity were measured by PicoGreen (Thermo Fisher Scientific, Waltham, MA, USA) and VICTOR Vivo Multimode Microplate Readers (PerkinElmer, Akron, OH, USA). For our study, the 18S rDNA V9 barcode was used, because it has often been applied to semi-quantitatively estimate relative abundances within a sample [31; 32; 33]. More specifically, we obtained the primer information from a study by [PERSON] et al. [33], which also followed the universal  
Figure 1: Map of the study sites (black closed circles) in Gwangyang Bay. primers for 18S V9 region designed by [PERSON] et al. [34]. The primer sequences are as follows: 18S V9 primer including adaptor sequence (Forward Primer: 5' TCGTCGGCAGCGTCAGATGTGT ATAAGAGACAGCCCTGCCHTTGTACACAC 3', Reverse Primer: 5' GTCTGGTGGCTCGGCA GATGGTATAAGAGACAGCCTTCYGCAGGTTCACCTAC 3', the primers are in bold). The PCR master mixture of 25 \(\upmu\)L (Macrogen Inc., Seoul, Korea) comprised 2 \(\upmu\)L of genomic DNA (1 ng/\(\upmu\)L), 1.25 \(\upmu\)L of each primer (5 \(\upmu\)M), 5 \(\upmu\)L of 5 \(\times\) Hercules II Reaction Buffer, 0.25 \(\upmu\)L of dNTP mix (100 mM), 0.5 \(\upmu\)L of Hercules II Fusion DNA polymerase (Agilent, Waldbronn, Germany), and 14.75 \(\upmu\)L of PCR Grade water. To amplify the target region attached with adapters, as a first PCR process, the extracted DNA was amplified by 18S V9 primers with one cycle of 3 min at 95 \({}^{\circ}\)C, 25 cycles of 30 s at 95 \({}^{\circ}\)C, 30 s at 55 \({}^{\circ}\)C, 30 s at 72 \({}^{\circ}\)C, and a final step of 5 min at 72 \({}^{\circ}\)C for amplicon PCR product. As a second process, to produce indexing PCR, the first PCR product was subsequently amplified with one cycle of 3 min at 95 \({}^{\circ}\)C, 8 cycles of 30 s at 95 \({}^{\circ}\)C, 30 s at 55 \({}^{\circ}\)C, 30 s at 72 \({}^{\circ}\)C, and a final step of 5 min at 72 \({}^{\circ}\)C. A subsequent limited-cycle amplification step was performed to add multiplexing indices and Illumina sequencing adapters (Figure 2). The final products were normalized and pooled using the PicoGreen (ThermoFisher Scientific, Waltham, MA, USA), and the size of the libraries was verified using the LabChip GX HT DNA High Sensitivity Kit (PerkinElmer, Waltham, MA, USA). A sequencing library is prepared by random fragmentation of the DNA or cDNA sample, followed by 5' and 3' adapter ligation. Alternatively, \"tagmentation\" combines the fragmentation and ligation reactions into a single step that greatly increases the efficiency of the library preparation process. Adapter-ligated fragments are then PCR amplified and gel purified. The PCR products were sequenced using the MiSeq(tm) platform (Illumina, San Diego, CA, USA) from commercial service (Macrogen Inc., Republic of Korea). In total, filtered 6,151,975 paired-end reads from the 30 samples were generated on the platform, of which 97.11% passed Q30 (Phred quality score > 30) in this study. Raw reads were trimmed with CD-HIT-OTU and chimeras were identified and removed using rDNAools. For paired-end merging, FLASH (Fast Length Adjustment of Short reads) version 1.2.11 was used. Each sample yielded paired-end reads ranging from 21,101-299,305 reads (mean: 180,940 reads), and all samples exhibited the saturation of the number of operational taxonomic units (OTUs) by rarefaction curve analysis (see Appendix A). Merged reads were processed and were clustered into OTUs using a bioinformatic algorithm, UCLUST [35], at a 97% OTU cutoff value (352 OTUs in gamma-diversity). The resulting 552 OTUs were classified into 19 genus-level taxonomic groups (those representing < 0.04% abundance were not plotted).