16S-Seq of sea water: Liaohe Estuary
The V3V4 regions of 16S rRNA genes from each extracted DNA sample were amplified using primers 341F-806R (341F: ACTCCTACGGGAGGCAGCAG, 806R: GGACTACHVGGGTWTCTAAT). All polymerase chain reactions (PCRs) were performed in 15 L reactions with 7.5 L Phusion High-Fidelity PCR Master Mix (New England Biolabs), 1 L of forward and reverse primers (10 M), 1 L of dNTPs (2.5 mM), and 1 L of template DNA. Thermal cycling consisted of initial denaturation at 95 C for 2 min, followed by 25 cycles of denaturation at 94 C for 5 s, annealing at 55 C for 30 s, and elongation at 72 C for 30 s. The PCR products of each sample were detected by electrophoresis on 1.5% agarose gel. The obtained PCR products for 16S and 18S rRNA genes were purified, quantified, and mixed in equal amounts to construct the sequencing libraries. Finally, the library quality was determined using the Agilent Bioanalyzer 2100 system and Qubit 2.0 Fluorometer (Thermo Scientic), and then all libraries were sequenced on an Illumina NovaSeq platform.