Archaeal 16S rRNA gene sequences from Guaymas Basin 50°C enrichment
DNA from AOM enrichments was extracted as described before (Zhou et al. 1996). The protocol encompassed three cycles of freezing and thawing, chemical lysis in a high-salt extraction buffer (1.5M NaCl) by heating of the suspension in the presence of sodium dodecyl sulfate and hexadecyltrimethyl-ammonium bromide, and treatment with proteinase K, followed by chloroform:isoamylalcohol extraction (24:1) and isopropanol based nucleic acid precipitation.To obtain an overview about the diversity of rare microbial phyla we performed massive parallel tag sequencing of ten long-term AOM enrichment cultures. In addition to the four enrichments presented in detail (E20, GB37, GB50 and GF) we also investigated six enrichment cultures that were inoculated with samples from Amon mud volcano, a Black Sea microbial reef, Black Sea sediments, Caldera mud volcano, the Gulf of Mexico and Hydrate Ridge. The latter six enrichment cultures were used for comparison, but are not focus of this study. From DNA that was extracted as described above, we amplified 16S rRNA genes using the primer pairs Arch20F/Arch958RV (Massana et al. 1997, Pires et al. 2012) Pires et al. 2012) for archaea. The amplicon libraries were prepared, and the V3-V5 region of these amplicons was sequenced on a 454 Genome Sequencer GS FLX+ (Roche, Basel, Switzerland) at the Max Planck Genome Centre (Cologne, Germany). The raw read data was processed based on a standard operating procedure (Schloss Patrick D et al. 2011) using mothur (release 1.33, 02/2014; (Schloss P. D. et al. 2009). Reads were denoised based on PyroNoise (Quince et al. 2009), trimmed, preclustered ((Huse et al. 2010)) and chimeras removed ((Edgar et al. 2011)