title: Methanogenic culture methagenome
design: The V1-V3 variable region of the 16S rRNA gene was amplified by PCR with universal primers, containing rRNA-specific parts: A2Fa (TCCGGTTGATCCTGCC)  and A519R (GTGCTCCCCCGCCAATTCCT). To generate FLX libraries, we used one-way reads amplicon sequencing approach recommended by Roche (see  GS Junior System Guidelines for Amplicon Experimental Design, Aug 2010 , Section 7). The forward amplicon primers contained FLX A-key sequence, multiplex identifier decanucleotide sequence and A2Fa 16S rRNAspecific sequence. The reverse primer contained FLX B-key sequence followed by A519R sequence. 16S rRNA amplicons were generated under the following conditions: 2 min at 96 C; 30 cycles of 30 s at 96 C, 45 s at 58 C and 40 s at 72 C, followed by 10 min at 72 C using PCR kit Amplisens (Interlabservice, Russia) on thermocycler  BIS M-111  (BIS-N, Novosibirsk, Russia). We used E.coli total DNA as template in positive control PCRs, and no DNA template were added into negative control PCR mixes. For each sample, four independent PCR reactions were performed in total volume 20 ul. The resulting PCRs were pooled and the target 16S rRNA fragments were sizeselected by electrophoresis in 0.8% agarose gel, followed by amplicon DNA extraction from the gel slice. The resulting amplicon library was used for pyrosequencing with the GS FLX 454 system (Roche, United States), according to the manufacturer s GS FLX Titanium Sequencing Method Manual at the Limnological Institute of the Siberian Branch of RAS. Sequencing was performed within the standard GS FLX run along with other amplicon libraries