title: 454 pyrosequencing for microbial community study
design: DNA extracts were amplified with: custom primers located at position 785-1061 of the E. coli 16S rRNA bacterial gene comprising the V5 and V6 hypervariable regions. The forward primer od each primer pair used holds a unique 10-base sample identification barcoding tags to separate samples after sequencing. Three PCR reactions were carried out for each sample and then pooled. PCR products were purified and quantified with Bioanalyser. Equivalent molarity of each of the amplicon-pools were bulked for the subsequent pyrosequencing with the 454 Life Sciences GS FLX system.