Pyrotag sequencing of methanogenic benzene-degrading cultures
In this experiment, the hypervariable V6 to V8 region of the 16S rRNA gene was amplified using PCR reactions on a PTC-200 thermal cycler (MJ Research Inc., Waltham, MA, USA) with general primer targeting 16SrRNA of bacteria, archaea and 18S rRNA of some eukaryotes at 926f/1392r (Engelbrektson et al., 2010; Hanshew et al., 2013). The forward primer contained a 30 bp adapter sequence (CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG). The reverse primer also contained a 30bp adapter sequence (CCT ATC CCC TGT GTG CCT TGG CAG TCT CAG), as well as a 10 bp multiplex identifier barcode (MID) to distinguish multiple samples pooled within one sequencing region. PCR reactions were prepared and conducted as described in Ramos-Padr—n et al. (2010b) except that 54_C was used as annealing temperature. The PCR products were purified using the GeneJETTM PCR Purification Kit (Fermentas, Thermo Scientific, Waltham, MA, USA) according to the manufacturerÕs instructions. The quality of cleaned PCR product was checked on a 2% agarose gel. The quantity of DNA was measured with a NanoDrop 1000 Spectrophotometer (Nanodrop, Wilmington, DE, USA). The purified and quantified PCR products were sent to the Genome Quebec and McGill University Innovation Center, where samples were checked for quality again, pooled, and processed using unidirectional sequencing (Lib-1 chemistry) using Roche GS FLX Titanium technology.