title: Bacterial en eukaryotic metacommunity analysis of inland icefree soils: Sample 11PER003
design: "A total of 22 samples were selected for Next generation sequencing analysis on a Roche 454 instrument. Extracellular DNA was removed following Corinaldesi (2005). Genomic DNA was isolated as described by Zwart (1998). This included a beat-beating method with phenol extraction and ethanol precipitation. After extraction, the DNA was purified on a Wizard column (Promega). For the prokaryotes, the V1 to V3 variable regions of the 16S rRNA were amplified using fusion primers containing the 454/Roche adaptor, a unique 454/Roche MID   and  the general forward prokaryote primer pA according to Edwards  (1989) or the general reverse prokaryote primer BKL1 (5’-GTATTACCGCGGCTGCTGGCA-3’), which resulted in a fragment of 557 bp. (Cleenwerck 2007). For the eukaryotes, the V4 region was amplified using a forward fusion primer containing the 454/Roche adaptor, a unique 454/Roche MID’s and a general forward eukaryote primer TAReuk454FWD1 (Stoeck 2010) and a reverse fusion primer with TAReukREV3 as the general eukaryotic primer (Stoeck 2010), resulting in a fragment of 446bp. The sequencing was only performed in the forward direction. The PCR amplification conditions, purification, quality control and bioinformatics analysis of the pyrosequences are described in Tytgat (2014). Using the Wang classifier algorithm (Wang 2007), the OTUs of the prokaryotes were mapped against the Greengenes database 13_5 (DeSantis 2006) and those of the eukaryotes against PR2 (version of May 2013) (Guillou 2013). "