16S rRNA of marine sediment
DNA was extracted using the POWERSOIL DNA isolation kit (MO BIO, Carlsbad, US) according to manufacturer guidelines. Barcoded 16S rRNA gene pyrosequencing was carried out on 9 samples according to Berry et al (2011). PCR primers targeting the bacterial 16S rRNA gene of most bacteria (909F:5- ACTCAAAKGAATWGACGG-3, 1492R:5-NTACCTTGTTACGACT-3) were used for the first PCR amplification step. Archaeal 16S rRNA sequences were first amplified using S-D-Arch-0025-a-S-17 (5-CTGGTTGATCCTGCCAG-3) forward primer and S-*-Univ-0907-a-A-20 (5-CCGTCAATTCMTTTRAGTTT-3) reverse primer (Vetriani et al. 1999). PCR reactions were conducted in 50 mL volume using 1.25U Pfu DNA polymerase (Fermentas), 1mM primers, 0.16 mM dNTPmix, 1x Pfu buffer, and template DNA. All PCR reactions were performed in triplicate and then pooled. The thermal program consisted of an initial 95C denaturation step for 4min, a cycling program (25cycles) of 95C for 30 s, 52C for 45 s, and 72C for 90 s, and a final elongation step at 72C for 7 min. For the second step, long oligonucleotides were used consisting of the gene-specific PCR primer sequences tagged with the sequencing adapters for GS FLX Titanium chemistry, which were designed as recommended by the manufacturer (454 Life Sciences) and according to Berry et al. (2011). The reverse primer (5-ACTCAAAKGAATWGACGG-3) included an 8bp barcode identifier (Hamady et al., 2008). The second step was carried out using the above thermal program but with 5 cycles. PCR reactions were performed in triplicate and then pooled to yield a final concentration of at least 250 g mL-1. For sequencing, PCR amplicons were purified using the Roche High Purity PCR purification kit and final DNA concentration measured using a Nanodrop1000 Spec UV/Vis spectrophotometer. Amplicons for pyrosequencing were submitted to the sequencing facility in the Department of Biochemistry, Cambridge University (UK) where pyrosequencing was performed using a Roche 454 Junior sequencer.16S rRNA gene sequences were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME, v1.8.0) as described by Caporaso et al. (2010). Sequences with quality scores lower than 25 were eliminated and sequences with 200600 bp were assigned to samples with 8 bp barcodes. Following removal of chimeras, sequences were clustered at 97% similarity into relevant OTUs using Usearch (Edgar, 2010). Representative OTU sequences were picked, and aligned using the PyNast algorithm (Caporaso et al. 2010; DeSantis et al., 2006) prior to taxonomy assignment using the GreenGenes (core13_8_2013) classifier at 97% identity Wang et al., 2007). Alpha diversity was estimated within QIIME by rarefaction curves (observed species metric), the Chao1 species richness index (Cha, 1984), and the Shannon diversity index (Shannon, 2001).