alias: NIOZ_Jetty_10012011
title: Microbial diversity in suspended particulate matter collected in the coastal North       Sea
design: 16S rRNA gene amplicon library with specific primers targeting bacteria         and archaea. PCR reactions were performed with the universal (Bacteria and Archaea) primers S-D-Arch-0519-a-S-15 (5’-CAG CMG CCG CGG TAA-3’) and S-D-Bact-785-a-A-21 (5’-GAC TAC HVG GGT ATC TAA TCC-3’) (Klindworth et al., 2013 Nucleic Acids Res 41:e1. doi: 10.1093/nar/gks808.) adapted for 454 pyrosequencing by the addition of sequencing adapters and multiplex identifier (MID) sequences. Each 30 µl PCR reaction comprised 5× Phusion HF Buffer containing 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 µM of each primer, 1 U Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Pittsburgh, PA). The following PCR conditions were used: initial denaturation at 98°C for 30 s, followed by 25 cycles consisting of denaturation (98°C for 10 s), annealing (53°C 20s), and extension (72°C 30s) and a final extension step at 72°C for 7 min. To minimize PCR bias three individual reactions were performed per template. PCR products were pooled, loaded in a 0.8% agarose gel and purified using Qiagen Qiaquick gel extraction kit (Qiagen, Germany). PCR products were quantified with the Quant-iT™ PicoGreen® dsDNA Assay Kit (Life technologies, Netherlands). Equimolar concentrations of the barcoded PCR products were pooled and sequenced on GS FLX Titanium platform (454 Life Sciences) by Macrogen Inc. Korea using the Lib_L chemistry from primer A side.
identifiers.namespace: Royal Netherlands Institute for Sea Research
identifiers.id: NIOZ_Jetty_10012011