Characterization of the infant GUT bifidobacterial population by 16S amplicon pool pyrosequencing
The PCR products deriving from the 16S rDNA hypervariable regions amplification were purified with Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers, then the purified amplicons were quantified by using the Quant-iT PicoGreen dsDNA kit (Invitrogen, Leek, Netherlands).  After the quantification step, the amplicons were pooled in equal amounts and fixed to micro beads to be clonally amplified by performing an emulsion PCR following the GS-FLX protocol Titanium emPCR LIB-A (454 LifeSciences, Roche, Branford, CT, USA). After the amplification the beads have been enriched in order to keep only those carrying identical PCR products on their surface, and then loaded onto a picotiter plate for the pyrosequencing reaction following the GS-FLX Titanium sequencing protocol.
Institute of Biomedical Technologies, National Res
Infant human GUT microbiota 13