PyroTag: Pyrosequencing as a Powerful Tool to Study the Diversity of Microbial Community

At JGI, major efforts have been spent on using the 16S RNA sequencing to study the composition of bacterial community. Currently, 16S rRNA genes from a microbial community are amplified with conserved PCR primers and the mixed amplicon is cloned and sequenced using traditional Sanger sequencing method. Using Roche GS FLX amplicon sequencing method, individual amplicon can be sequenced directly on the platform, producing hundreds of thousand ~;250 bp tags of the full length 16S rRNA gene. We call this pipeline PyroTag. The benefit of the PyroTag technology includes higher resolution and less bias than traditional PCR and cloning based sequencing. The disadvantage is that the read length is short as we used FLX reagent kit for the study. The aim for this study is to systematically test the effect of amplicon length and 16S rRNA regions targeted on diversity coverage of complex microbial community. The goal is to develop a standard PyroTag pipeline to provide to broad range JGI users. Molecular barcode method has been used to distinguish different amplicon length and different targeted 16S regions. Strategy and detailed experiment design as well as preliminary results will be presented