16S rRNA amplicon sequencing of subantarctic seawater: sub-surficial sample station A, february
Samples were sequenced at INDEAR (Argentina). Quantification of initial DNA samples was performed using the Quant-iTTM PicoGreen DNA Assay Kit (Invitrogen) according to manufacturers instructions (high range curve - medium area plate).For the preparation of the libraries, 10 ng of DNA were used. A 1st PCR allowed the amplification of the region of interest obtaining an amplicon of 400 bp. Then the samples were purified and a second round of amplification allowed to identify each one of them by adding an index. After the 2nd PCR, samples were purified and the average final amplicon size was checked to be  400 bp. The samples were then quantified again using the Quant-iTTM PicoGreen . Libraries were pooled at the same concentration, 50 ng / ul. To check for quality, 1ul of the pool was run in the 2100 Bioanalyzer (Agilent Technologies) using the DNA 12000 chip Kit. The library pool was quantified using DeNovix, according to the instructions detailed by the manufacturer (high range curve - half area plate). From the estimated concentration values, samples were normalized to 3.68 nM and used for the generation of clusters in the lane of the sequencing cell. Samples were pooled at 75% and the remaining 25% of the lane was filled with PhiX. Libraries were placed in the lane of the sequencing cell and sequencing runs were performed generating paired ends (PE) 2x150bp reads.