The 16S rRNA phylogeny in the surface of upwelling water induced by the intrusion of Kuroshio Current
Approximately 50 ng of environmental DNA collected from the surface of upwelling water was used as the template for polymerase chain reaction (PCR) to specifically amplify the hypervariable V6-V9 region of 16S rRNA genes using the high-fidelity Phusion DNA Polymerase (New England Biolabs, Massachusetts, USA) and the primer set which is 16S-909F (5’-ACTCAAAKGAATWGACGG-3’) and 16S-1492R (5’-NTACCTTGTTACGACT-3’). Equimolar amounts of amplicons from two libraries were pooled for sequencing on a Roche 454 Genome Sequencer FLX+ Instrument (454 Life Sciences, Connecticut, USA).