title: Anaerobic digesters Metagenome
design: DNA was extracted from 0.30 g sludge material using PowerSoil DNA isolation kit (Mo Bio, Carlsbad, CA, USA). Fragments of approximately 250 bp spanning the V4 region of 16S ribosomal gene was amplified by PCR using the universal primer set F515/R806, containing Roche 454 pyrosequencing adapters and sample-specific MID barcodes in between the adapter and the forward primer. Amplicons within the appropriate size range were gel-purified and quantified using the Qubit fluorometer with the high-sensitivity DNA reagent kit (Invitrogen, Carlsbad, CA, USA), and amplicons were pooled in equimolar quantities (1.00*10^9 molecules/μl). The quality of the libraries was assessed using a Bioanalyzer 2100 with high-sensitivity chip (Agilent Technologies, Waldbronn, Germany). Each amplicon library was sequenced on a separate 1/8th Pico Titer Plate (PTP) section using GS-FLX instrument and Titanium chemistry (Roche Applied Science, Mannheim, Germany).