Reshaping the Gut Microbiome with Bacterial Transplantation and Antibiotic Intake
In order to analyze bacterial composition, the V4 hypervariable region of the 16S gene was amplified from the DNA extracted from each sample using two universal primers: V4F_517_17 (5&apos;-GCCAGCAGCCGCGGTAA-3&apos;) and V4R_805_19 (5&apos;-GACTACCAGGGTATCTAAT-3&apos;). Each of the multiplex identifiers (MIDs) has been added upstream the forward primer sequence (V4F_517_17). Standard PCR was run in a Mastercycler gradient (Eppendorf, Madrid, Spain) at 94 degrees C for 2 min followed by 35 cycles of 94 degrees C for 30 s, 56 degrees C for 20 s, 72 degrees C for 40s and a final cycle of 72 degrees C for 7 min. Ten of twelve MIDs proposed by Roche resulted in a good amplification of the PCR product. The 16S rRNA V4 amplicons were subsequently sequenced on a 454 Genome Sequencer FLX platform (Center for Genomic Regulation, Barcelona, Spain) according to 454 platform protocols.