title: Obere Hollacke_16S_2018
design: Prokaryotic 16S rRNA gene amplification was carried out using the following primer pairs: EMBf 515F (GTGYCAGCMGCCGCGGTAA, Parada et al., 2016)  EMBr 806R (GGACTACNVGGGTWTCTAAT, Apprill et al., 2015) for the V4 region of the prokaryotic 16S rRNA gene elongated with sequencing barcodes and adapters. Amplification of the target taxonomic marker gene regions and sequencing were carried out by LGC Genomics (Berlin, Germany). The PCRs included about 1-10 ng of DNA extract (total volume 1l), 15 pmol of each forward primer and reverse primer in 20 L volume of 1 x MyTaq buffer containing 1.5 units MyTaq DNA polymerase (Bioline GmbH, Luckenwalde, Germany) and 2 l of BioStabII PCR Enhancer (Sigma-Aldrich Co.).Amplification was carried out for 30 cycles using the following parameters: 1 min 96C pre-denaturation; 96 C denaturation for 15 s, 55 C annealing for 30 s, 70 C extension for 90 s, hold at 8 C. The amplicon pools were purified with one volume Agencourt AMPure XP beads (Beckman Coulter, Inc., IN, USA) to remove primer dimer and other small mispriming products, followed by an additional purification on MiniElute columns (QIAGEN GmbH, Hilden, Germany). About 100 ng of each purified amplicon pool DNA was used to construct Illumina libraries using the Ovation Rapid DR Multiplex System 1-96 (NuGEN Technologies, Inc., CA, USA). Illumina libraries (Illumina, Inc., CA, USA) were pooled and size selected by preparative gel electrophoresis. Sequencing was performed on an Illumina MiSeq using 300 bp paired-end format with a V3 Reagent Cartridge on the Illumina MiSeq platform, aiming for 50,000 raw sequence read pairs per sample. Demultiplexing of libraries for each sequencing lane was carried out using the Illumina bcl2fastq 2.17.1.14 software and sequencing adapters were removed from the end of the sequences.