title: Anaerobic digesters metagenome
design: For pyrosequencing, 16S rRNA genes were amplified from extracted DNA using primer sets 341F/805R, where an adapter sequence and a unique identifier tag sequence were fused upstream the forward primer (Table S1). Duplicate PCR amplifications were conducted on each duplicate DNA extract in a 20 μl reaction volume containing 0.5 µM of each primer, 0.15 mM of each dNTP, 1x Titanium Taq PCR buffer, 1U Titanium Taq DNA polymerase (Clontech, Palo Alto, CA, USA), and 1 µl genomic DNA. Reactions were performed on a T100 thermal cycler (Bio-Rad, Hercules, CA, USA) under following conditions: 94°C for 10 min (1); 94°C for 45 s, 61°C (bacteria) or 63°C (Archaea) for 45 s, 72°C for 45 s (35); 72°C for 10 min (1). The duplicate PCR amplifications were pooled and following gel electrophoresis amplicons within the appropriate size range were gel-purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). Purified amplicons were quantified using the Qubit fluorometer (Invitrogen, Carlsbad, CA, USA) and diluted to equal quantities. An additional quantification was conducted with qPCR analyses with primer set targeting the fusion primer adapters located at the 5 end of the amplicon. The 20 µl qPCR reaction mixture consisted of 0.5 µM of each primer (emPCR, Table S2) and 10 µl iTaq universal SYBR Green supermix (2x) (Bio-Rad Laboratories, Hercules, CA, USA). Cycling was performed at 95°C for 2 min (1), 95°C for 15 s, annealing at 58°C for 30 s and 60°C for 30 s (40) using the Applied Biosystems StepOnePlus real-time PCR system (Applied Biosystems, Waltham, MA, USA) and software version 2.2 (Applied Biosystems, Waltham, MA, USA). At the end of each qPCR assay a temperature melt curve was performed to verify reaction quality (60-95°C, ΔT per 15 s = 0.3°). The barcoded amplicons were pooled in equimolar quantities in order to process for sequencing (Roche GS-FLX+ Titanium System), performed according to the manufacturer’s instructions (Roche Applied Science, Mannheim, Germany).