Metegenome assesment at ending time point in biogas fermentor containing casein substrate
DNA quality and quantity measurements were performed on Bioanalyzer (Agilent Technologies) and Qubit (Life Technologies). Total DNA samples from three replicates were pooled and processed using the SOLiD Fragment-Seq Kit (Life Technologies), according to the manufacturers suggestions. Briefly, 5 ug of metagenome DNA was fragmented, the 50-200nt fraction was size selected, sequencing adaptors ligated. The DNA fragment library was purified with Qiagen MinElute PCR Purification Kit (Qiagen) and size-selected on a 6% TBE-Urea denaturing polyacrylamide gel. The 150-250nt DNA fraction was amplified using AmpliTaq polymerase and purified by AmPureXP Beads. Concentration of each library was determined using the SOLiD Library TaqMan Quantitation Kit (Life Technologies). Each library was clonally amplified on SOLiD P1 DNA Beads by emulsion PCR (ePCR). Emulsions were broken with butanol, and ePCR beads enriched for template-positive beads by hybridization with magnetic enrichment beads. Template-enriched beads were extended at the 3' end in the presence of terminal transferase and 3' bead linker. Beads with the clonally amplified DNA were deposited onto sequencing slide and sequenced on SOLiD V4 Instrument using the 50-base sequencing chemistry.