VX765 The S PCR libraries were generated for the samples
The 16S PCR libraries were generated for the 5 samples. The primers E9–29 and E514–530 (Brosius et al., 1981), specific to bacteria, were selected for their theoretical ability to generate the least bias of amplification capability among the various bacterial phyla (Wang and Qian, 2009). The oligonucleotide design includes 454 Life Sciences A or B sequencing titanium adapters (Roche Diagnostics Belgium NV, Vilvoorde, Belgium) and multiplex identifiers fused to the 5′ end of each primer. The amplification mix contains 5 U of FastStart highfidelity polymerase (Roche Diagnostics Belgium NV), 1× enzyme reaction buffer, 200 μM deoxynucleotide triphosphates (dNTP; Eurogentec SA, Liege, Belgium), 0.2 μM concentration of each primer, and 100 ng of genomic DNA in VX765 volume of 100 μl. Thermocycling conditions consisted of a denaturation step at 94 °C for 15 min, followed by 25 cycles of 94 °C for 40 s, 56 °C for 40 s, 72 °C for 1 min, and a final elongation step of 7 min at 72 °C. These amplifications were performed on an EP Master system gradient apparatus (Eppendorf AG, Hamburg, Germany). The PCR products were run on a 1% agarose electrophoresis gel and the DNA fragments were extracted and purified using an SV PCR purification kit (Promega Benelux B.V., Leiden, The Netherlands). The quality and quantity of the products were assessed using a PicoGreen double-stranded DNA (dsDNA) quantitation assay (Isogen Life Science NV). All libraries were run in the same titanium pyrosequencing reaction using Roche multiplex identifiers. All amplicons were sequenced using the Roche GS-Junior Genome Sequencer instrument (Roche Diagnostics Belgium NV).