nifH gene amplicon sequence in filtered seawater
Between 7.5 and 10 L of surface seawater per replicate (4 replicates) were collected and filtered using silicone tubes and peristaltic pumps through sterile STERIVEX 0.22 m pore size filters (Millipore, USA). Summer samples of July 2018 were prefiltered with a 200 m mesh filter. Filters were preserved with 1.8 mL of lysis buffer (50 mM Tris-HCl pH 8.3, 40 mM EDTA pH 8.0, 0.75 M sucrose), immersed in liquid nitrogen and subsequently stored at 80 C until further analysis. DNA was extracted using the PowerWater DNA Isolation Kit (Mobio, Carlsbad, CA, USA.), quantified and quality-checked (according to the A260/A280 ratio) using a spectrophotometer NanoDrop 2000TM (Thermo Fisher Scientific). Amplification of nifH gene and sequencing of the amplicons were performed following Zehr & McReynolds, 1989. The PCR reactions were run on a T100TM Thermal cycler (BIO-RAD). The amplified products were purified using PCR Extract Mini Kit (5PRIME) and quantified using a spectrophotometer NanoDrop 2000TM (Thermo Fisher Scientific). Prior to sequencing, an additional PCR amplification of 10 cycles with custom barcoded nifH1 and nifH2 primers was carried out. After purification and library preparation from the barcoded PCR products, paired-end sequencing was performed using the MiSeq reagent kit with V2 chemistry (500 cycles) at the facilities of IMGM Laboratories GmbH (Germany) on the Illumina MiSeq Next Generation Sequencing technology (Illumina Inc.). The resulting 2x250 base pairs (bp) reads were demultiplexed on the instrument.