AMPLICON sequencing of seawater genomic
Genomic DNA of the pico- and nanoplankton was extracted using FastDNA SPIN Kit according to the manufacturers instructions (MP Biomedicals, Santa Ana, CA, USA). The integrity of the extracted DNA was assessed by gel electrophoresis, and the concentration and quality of DNA were measured with a spectrophotometer Nanodrop 2000c (Thermo-Fisher, USA). The V4 regions of bacterial 16S rRNA gene and eukaryotic 18S rRNA gene were amplified using PCR with the primer sets 515F (5-GTGCCAGCMGCCGCGG-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3), and 528F (5-GCGGTAATTCCAGCTCCAA-3) and 706R (5-AATCCRAGAATTTCACCTCT-3), respectively. For multiplexing of the samples in high-throughput sequencing, the forward bacterial and eukaryotic primers were inserted with 6-bp barcodes. The 20-L reaction solution contained Phusion High-Fidelity PCR Master Mix with GC Buffer (New England Biolabs, Ipswich, MA, USA), 1 L DNA template and 0.2 M of each primer. The PCR amplification conditions were as follows: 98 C for 1 min, followed by 30 cycles of denaturation at 98 C for 10 s, annealing at 50 C for 30 s, and extension at 72 C for 30 s, with a final extension at 72 C for 5 min. The PCR products were purified with a GeneJET Gel Extraction Kit (Thermo Scientific), and the libraries were generated using Illumina TruSeq DNA PCR-Free Library Preparation Kit (Illumina, USA). Sequencing was conducted on an Illumina MiSeq PE385 platform (Novogene, Beijing, China).